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EP4305070A1 - Modulation de la signalisation wnt dans des troubles gastro-intestinaux - Google Patents

Modulation de la signalisation wnt dans des troubles gastro-intestinaux

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Publication number
EP4305070A1
EP4305070A1 EP22767927.1A EP22767927A EP4305070A1 EP 4305070 A1 EP4305070 A1 EP 4305070A1 EP 22767927 A EP22767927 A EP 22767927A EP 4305070 A1 EP4305070 A1 EP 4305070A1
Authority
EP
European Patent Office
Prior art keywords
wnt
engineered
cells
tissue
wnt agonist
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22767927.1A
Other languages
German (de)
English (en)
Inventor
Russell FLETCHER
Sungjin Lee
Yang Li
Chenggang LU
Parthasarathy SAMPATHKUMAR
Geertrui VANHOVE
Wen-Chen Yeh
Liqin XIE
Leonard Presta
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Surrozen Operating Inc
Original Assignee
Surrozen Operating Inc
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Filing date
Publication date
Application filed by Surrozen Operating Inc filed Critical Surrozen Operating Inc
Publication of EP4305070A1 publication Critical patent/EP4305070A1/fr
Pending legal-status Critical Current

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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0679—Cells of the gastro-intestinal tract
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C—CHEMISTRY; METALLURGY
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    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • C07K2317/526—CH3 domain
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/71—Decreased effector function due to an Fc-modification
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    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled

Definitions

  • This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in txt format.
  • the .txt file contains a sequence listing entitled SRZN_020_03WO_ST25.txt created on March 7, 2022 and having a size of 80 kilobytes.
  • the sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.
  • the disclosure provides WNT signal modulators as a treatment for gastrointestinal disorders, in particular, inflammatory bowel diseases.
  • WNT proteins form a family of highly conserved secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis. WNT genes and WNT signaling are also implicated in cancer Insights into the mechanisms of WNT action have emerged from several systems: genetics in Drosophila and Caenorhabditis elegans ; biochemistry in cell culture and ectopic gene expression in Xenopus embryos. Many WNT genes in the mouse have been mutated, leading to very specific developmental defects. As currently understood, WNT proteins bind to receptors of the Frizzled family on the cell surface.
  • the signal is transduced to beta-catenin, which then enters the nucleus and forms a complex with TCF to activate transcription of WNT target genes.
  • beta-catenin a complex with TCF to activate transcription of WNT target genes.
  • Expression of WNT proteins varies, but is often associated with the developmental process, for example in embryonic and fetal tissues.
  • Dkkl Dickkopf-1
  • Mahl has been recently identified as the founding member of a family of secreted proteins that potently antagonize WNT signaling (see Glinka et al. (1998) Nature 391:357-62; Fedi et al. (1999) J Biol Chem 274: 19465-72; and Bafico et al. (2001) Nat Cell Biol 3:683-6).
  • Dkkl associates with both the WNT co-receptors, LRP5 and LRP6, and the transmembrane protein Kremen, with the resultant ternary complex engendering rapid LRP6 internalization and impairment of WNT signaling through the absence of functional Frizzled/LRP6 WNT receptor complexes (see, e.g., Mao et al. (2001) Nature 411:321-5; Semenov et al. (2001) Curr Biol 11:951-61; and Mao et al. (2002) Nature 417:664-7).
  • the adult intestinal epithelium is characterized by continuous replacement of epithelial cells through a stereo-typed cycle of cell division, differentiation, migration and exfoliation occurring during a 5-7 day crypt-villus transit time.
  • the putative growth factors regulating proliferation within the adult intestinal stem cell niche have not yet been fully identified, although studies have implicated the cell-intrinsic action of b-catenin/Lef/Tcf signaling within the proliferative crypt compartment.
  • IBD Inflammatory bowel disease
  • Crohn's disease and ulcerative colitis are the best-known forms of IBD, and both fall into the category of "idiopathic” inflammatory bowel disease because the etiology for them is unknown.
  • Active IBD is characterized by acute inflammation.
  • Chironic IBD is characterized by architectural changes of crypt distortion and scarring. Crypt abscesses can occur in many forms of IBD.
  • Crohn's disease can involve any part of the GI tract, but most frequently involves the distal small bowel and colon.
  • Inflammation is typically transmural and can produce anything from a small ulcer over a lymphoid follicle (aphthoid ulcer) to a deep fissuring ulcer to transmural scarring and chronic inflammation.
  • lymphoid follicle aphthoid ulcer
  • granulomas and extracolonic sites such as lymph nodes, liver, and joints may also have granulomas.
  • the transmural inflammation leads to the development of fistulas between loops of bowel and other structures. Inflammation is typically segmental with uninvolved bowel separating areas of involved bowel. The etiology is unknown, though infectious and immunologic mechanisms have been proposed.
  • Ulcerative colitis involves the colon as a diffuse mucosal disease with distal predominance.
  • the rectum is virtually always involved, and additional portions of colon may be involved extending proximally from the rectum in a continuous pattern.
  • the etiology for UC is unknown Patients with prolonged UC are at increased risk for developing colon cancer. Patients with UC are also at risk for development of liver diseases including sclerosing cholangitis and bile duct carcinoma.
  • all therapeutics in the clinic and most in development for treatment of UC focus on reducing inflammation and do not directly induce epithelial healing, highlighting the unmet need for therapeutic agents that promote epithelial repair.
  • WNT agonists for the regulation of intestinal epithelium growth is of great interest for clinical purposes.
  • exploration of WNT agonists as pharmacological agents has been hampered, in part, by the fact that they are not naturally soluble, diffusible molecules.
  • the present disclosure provides methods and compositions to specifically modulate WNT signaling through particular FZD receptors using engineered soluble WNT agonists.
  • engineered WNT agonists may achieve, for example, epithelial- specific transient Wnt signaling activation, which drives robust epithelial regeneration and barrier restoration, ultimately leading to a reduction in inflammation and amelioration of colitis.
  • the present disclosure provides engineered WNT agonist and related pharmaceutical compositions and methods of use.
  • the disclosure includes an engineered WNT agonist comprising: (a) one or more binding domains that bind to one or more FZD; and (b) one or more binding domains that bind to LRP5, LRP6, or both LRP5 and LRP6, wherein the engineered WNT agonist comprises a polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 1-18, or a polypeptide sequence disclosed in any one of SEQ ID NO:s 1-25, Figure 2, Figure 6, Table 1, or Table 3, or a functional fragment or variant thereof, e g., a binding fragment thereof, e.g., a VHH domain, a variable domain of a heavy chain, or a variable domain of a light chain.
  • the engineered WNT agonist comprises a polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 1-18, or a polypeptide sequence disclosed
  • the one or more binding domains that bind to one or more FZD bind to: i) FZD5; ii) FZD 8; iii) FZD 1; iv) FZD 2; v) FZD 7; vi) FZD 5 and FZD 8; vii) FZD 1, FZD 2, and FZD 7; viii) FZD 1, FZD 2, FZD 7, FZD 5 and FZD 8; ix) FZD4; x) FZD9; or xi) FZD10.
  • the engineered WNT agonist comprises one or more (e.g., two) polypeptide sequence having at least 90%, at least 95%, %, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:l-18 or 19-25 or a sequence disclosed in Table 3.
  • the engineered WNT agonist comprises: (a) one or more (e.g., two) polypeptide sequence having least 90%, or at least 95% homology to SEQ ID NO: 1 and one or more (e.g., two) polypeptide sequence having at least 90%, or at least 95% homology to SEQ ID NO:2; (b) one or more (e.g., two) polypeptide sequence having least 90%, or at least 95% homology to SEQ ID NO: 3 and one or more (e.g., two) polypeptide sequence having at least 90%, or at least 95% homology to SEQ ID NO:4; (c) one or more (e.g., two) polypeptide sequence having at least 80%, at least 90%, or at least 95% homology to SEQ ID NO: 5 and one or more (e.g., two) polypeptide sequence having at least 80%, at least 90%, or at least 95% homology to SEQ ID NO: 6; (d) one or more (e.g., two)
  • polypeptide sequence having at least 90%, or at least 95% homology to SEQ ID NO: 10 ; (f) one or more (e.g., two) polypeptide sequence having at least 90%, or at least 95% homology to SEQ ID NO: 7 and one or more (e.g., two) polypeptide sequence having at least 90%, or at least 95% homology to SEQ ID NO:8 (g) one or more (e.g., two) polypeptide sequence having at least 90%, or at least 95% homology to SEQ ID NO: 11 and one or more (e.g.
  • the polypeptide comprises the CDRs present in any one of SEQ ID NOs: 1 - 18 or 19-25.
  • the one or more binding domains that bind to LRP5, LRP6, or both LRP5 and LRP6 are humanized.
  • the engineered WNT agonists comprise a modified Fc domain, wherein the modified Fc domain comprises a LALAPG or N297G modification.
  • the WNT agonist has any of the structures or formats disclosed herein, including any of the various antibody-related structures or formats. Examples of suitable formats include, but are not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multi-specific antibodies ( e.g ., bispecific antibodies), and antibody fragments including but not limited to scFv, Fab, and Fab2, so long as they exhibit the desired biological activity, e.g., WNT agonist activity.
  • the WNT agonist is R2M13-h26.
  • R2M13 is a humanized form of the parental R2M13-26 that also comprises the LALAPG substitution in the Fc domain.
  • R2M13-h26 may also be referred to herein as R2M13-h26-LALAPG, R2M13-26 humanized LALAPG, or humanized LALPG.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an engineered WNT agonist disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the disclosure provides a method of treating a disease or disorder amenable to treatment by increased WNT pathway signaling in a subject, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein.
  • the disease or disorder is a gastrointestinal disorder, such as an inflammatory bowel disease.
  • the disease or disorder is selected from the group consisting of: Crohn’s disease (CD), CD with fistula formation, and ulcerative colitis (UC).
  • the engineered WNT agonist is administered orally or parenterally, e.g., intravenously, intraperitoneally, or subcutaneously.
  • the WNT agonist is R2M13-h26.
  • the WNT agonist is administered intravenously, e.g., as a bolus injection. In particular embodiments, the WNT agonist is administered at least once per week. In particular embodiments, the subject is administered about 0.5 to about 100 mg/kg body weight of the WNT agonist, or about 2 to about 50 mg/kg body weight of the WNT agonist, e.g., about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg.
  • the subject is administered about 3 to about 30 mg/kg body weight intravenously at least once per week of R2M13-h26, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO:9 and two polypeptides of SEQ ID NO: 10 bound by disulfide bonds.
  • the disclosure provides a method of increasing WNT signaling in a cell, comprising contacting the cell with an engineered WNT agonist disclosed herein.
  • the WNT agonist is R2M13-h26.
  • the disclosure provides a method of modulating expression of a WNT pathway molecule in one or more tissues and/or cells in a subject having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or the pharmaceutical composition disclosed herein.
  • the WNT pathway molecule is a gene or protein listed in any one of Tables 4-7.
  • the WNT pathway molecule is selected from the group consisting of: RNAse4, Angiogenin, Gsta3, Rnf43, Axin2, , or any of the genes or proteins listed in Table 7.
  • expression of the WNT pathway molecule is increased by at least 20%, at least 50%, at least 80%, at least 1.1 -fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, two-fold, at least five-fold, at least 10-fold, or at least 20-fold or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in one or more tissues and/or cells of the subject following administration of the engineered Wnt agonist.
  • the tissue is epithelial tissue.
  • the cells are gastrointestinal epithelial cells, optionally: stem cells, TA1, TA2, basal goblet cells, injury- induced alternative progenitors (AltEnteroPC), injury -induced alternative enterocytes (AltEntero), enterocyte precursors (EnteroPrecur), goblet cells 1, goblet cells 2, or enteroendocrine or tuft cells.
  • the WNT agonist is R2M13-h26.
  • the WNT agonist is administered intravenously, e.g., as a bolus injection.
  • the WNT agonist is administered at least once per week.
  • the subject is administered about 0.5 to about 100 mg/kg body weight of the WNT agonist, or about 2 to about 50 mg/kg body weight of the WNT agonist, e.g. , about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg.
  • the subject is administered about 3 to about 30 mg/kg body weight intravenously at least once per week of R2M13-h26, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO:9 and two polypeptides of SEQ ID NO: 10 bound by disulfide bonds.
  • the disclosure provides a method of stimulating tissue repair in a subject having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or the pharmaceutical composition disclosed herein.
  • the tissue repair is stimulated by (or the method results in) modulation of at least one WNT pathway molecule selected from the group consisting of: genes associated with the cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and/or any of the genes listed in any of Tables 4-8.
  • the genes associated with the cell cycle are selected from those provided in Table 4, or Aurka, Aurkb, Ccna2, Ccnbl, Ccnb2, Ccnd2, Ccnel, Cdc45, Cdkl, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plkl, Rrml, and Rrm2.
  • the genes associated with stem and progenitor cell renewal and differentiation are selected from those provided in Table 8, and Axin2, Idl, Hmga2, Nhp2, Foxql, and Adhl.
  • the genes associated with epithelial cell repair and barrier restoration are selected from those provided in Table 6, or Apexl, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zgl6, and Sprr2a3.
  • expression of the gene is increased by at least 20%, at least 50%, at least 80%, at least two-fold, at least five-fold, at least 10-fold, or at least 20-fold or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in one or more tissues and/or cells of the subject following administration of the engineered Wnt agonist.
  • the WNT agonist is administered intravenously, e.g., as a bolus injection. In particular embodiments, the WNT agonist is administered at least once per week. In particular embodiments, the subject is administered about 0.5 to about 100 mg/kg body weight of the WNT agonist, or about 2 to about 50 mg/kg body weight of the WNT agonist, e.g., about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg.
  • the subject is administered about 3 to about 30 mg/kg body weight intravenously at least once per week of R2M13-h26, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO:9 and two polypeptides of SEQ ID NO: 10 bound by disulfide bonds.
  • the disclosure provides a method of reducing inflammation in a subject having a gastrointestinal disorder (or a tissue or cells thereof), comprising administering to the subject an engineered WNT agonist or the pharmaceutical composition disclosed herein.
  • the inflammation is reduced by (or the method results in) modulation of at least one WNT pathway molecule selected from the group consisting of: genes provided in Table 5, or Adamdecl, Atf3, Gpx2, Gsta3, Gstml, Gstm3, Gdfl5, Ihh, 1118, Lyz2, Noxl, Reg4, Sycn, Selenbpl, Tgfbr2, and Timp3.
  • the inflammation is reduced in gastrointestinal tissue, optionally epithelial tissue
  • the inflammation is reduced in gastrointestinal epithelial cells, epithelial stem cells, TA1, TA2, basal goblet cells, injury-induced alternative progenitors (Alt progenitors), injury-induced alternative enterocytes (Alt Enterocytes), enterocyte precursors (EnteroPrecur), goblet cells 1, goblet cells 2, or enteroendocrine or tuft cells.
  • expression of the WNT pathway molecule is increased by at least 20%, at least 50%, at least 80%, at least 1.1 -fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, two-fold, at least Five-fold, at least 10-fold, or at least 20-fold or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in one or more tissues and/or cells of the subject following administration of the engineered Wnt agonist.
  • the WNT agonist is administered intravenously, e.g., as a bolus injection.
  • the WNT agonist is administered at least once per week.
  • the subject is administered about 0.5 to about 100 mg/kg body weight of the WNT agonist, or about 2 to about 50 mg/kg body weight of the WNT agonist, e.g., about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg.
  • the subject is administered about 3 to about 30 mg/kg body weight intravenously at least once per week of R2M13-h26, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO:9 and two polypeptides of SEQ ID NO: 10 bound by disulfide bonds.
  • the engineered Wnt agonist is R2M13-h26 or comprises a functional variant or fragment thereof.
  • the subject is a mammal, optionally a human.
  • the disclosure provides a method of restoring gastrointestinal epithelial barrier in a subjecting having injured epithelium, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein.
  • the WNT agonist is administered intravenously, e.g., as a bolus injection.
  • the WNT agonist is administered at least once per week.
  • the subject is administered about 0.5 to about 100 mg/kg body weight of the WNT agonist, or about 2 to about 50 mg/kg body weight of the WNT agonist, e.g., about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg.
  • the subject is administered about 3 to about 30 mg/kg body weight intravenously at least once per week of R2M13-h26, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO: 9 and two polypeptides of SEQ ID NO: 10 bound by disulfide bonds.
  • the gastrointestinal epithelial barrier is restored by modulation of at least one WNT pathway molecule selected from the group consisting of: genes associated with the cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and/or any of the genes listed in any of Tables 4, 5, 6, 7, 8, and 11.
  • the genes associated with the cell cycle may be selected from those provided in Table 4, or Aurka, Aurkb, Ccna2, Ccnbl, Ccnb2, Ccnd2, Ccnel, Cdc45, Cdkl, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plkl, Rrml, and Rrm2.
  • the genes associated with stem and progenitor cell renewal and differentiation may be selected from those provided in Table 8, and Axin2, Idl, Hmga2, Nhp2, Foxql, and Adhl.
  • genes associated with epithelial cell repair and barrier restoration may be selected from those provided in Table 6, or Apexl, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zgl6, and Sprr2a3.
  • the gastrointestinal epithelial barrier is restored by modulation of at least one WNT pathway molecule, wherein expression of the WNT pathway molecule is increased by at least 20%, at least 50%, at least 80%, at least 1.1-fold, at least 1.2- fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least two-fold, at least five-fold, at least 10-fold, or at least 20-fold or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in one or more tissues and/or cells of the subject following administration of the engineered Wnt agonist.
  • the gastrointestinal epithelial barrier is restored by modulation of at least one WNT pathway molecule, wherein expression of the WNT pathway molecule is increased in one or more tissues and/or cells of the subject within about 24 hours of administering the engineered Wnt agonist.
  • the subject’s injured epithelium is substantially restored within about 6 days of administering the engineered Wnt agonist.
  • administration of the engineered Wnt agonist to the subject does not induce over proliferation of normal epithelium.
  • the disclosure provides a method of inducing epithelial progenitor cell differentiation in a subject having a gastrointestinal disorder, comprising administering to the subject the engineered WNT agonist an engineered WNT agonist or the pharmaceutical composition disclosed herein.
  • the WNT agonist is administered intravenously, e.g., as a bolus injection.
  • the WNT agonist is administered at least once per week.
  • the subject is administered about 0.5 to about 100 mg/kg body weight of the WNT agonist, or about 2 to about 50 mg/kg body weight of the WNT agonist, e.g., about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg.
  • the subject is administered about 3 to about 30 mg/kg body weight intravenously at least once per week of R2M13-h26, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO:9 and two polypeptides of SEQ ID NO: 10 bound by disulfide bonds.
  • the epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule selected from the group consisting of: genes associated with the cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and/or any of the genes listed in any of Tables 4, 5, 6, 7, 8, and 11.
  • the genes associated with the cell cycle may be selected from those provided in Table 4, or Aurka, Aurkb, Ccna2, Ccnbl, Ccnb2, Ccnd2, Ccnel, Cdc45, Cdkl, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plkl, Rrml, and Rrm2.
  • the genes associated with stem and progenitor cell renewal and differentiation may be selected from those provided in Table 8, and Axin2, Idl, Hmga2, Nhp2, Foxql, and Adhl.
  • genes associated with epithelial cell repair and barrier restoration may be selected from those provided in Table 6, or Apexl, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zgl6, and Sprr2a3.
  • the epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule, wherein expression of the WNT pathway molecule is increased by at least 20%, at least 50%, at least 80%, at least 1.1 -fold, at least 1 ,2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, two-fold, at least five-fold, at least 10-fold, or at least 20-fold or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in one or more tissues and/or cells of the subject following administration of the engineered Wnt agonist.
  • the epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule, wherein expression of the WNT pathway molecule is increased in one or more tissues and/or cells of the subject within about 24 hours of administering the engineered Wnt agonist.
  • administration of the engineered Wnt agonist induces progenitor cell differentiation into enterocytes, goblet cells, enteroendocrine, or tuft cells in the subject. In some embodiments, substantial progenitor cell differentiation is induced in the subject within about 48 hours of administering the engineered Wnt agonist. In some embodiments, administration of the engineered Wnt agonist to the subject does not induce over proliferation of normal epithelium.
  • Figure 1 provides an illustrative structure of one embodiment of an engineered WNT agonist.
  • the R2M13 anti-Fzd5,8 antibody includes two heavy chains and two light chains, and each light chain also includes an anti-LRP6 VHH fused to its N-terminus via a tag.
  • Figure 2A provides an amino acid sequence alignment of the parental LRP6 binding VHH, VHH26, and the closest human germline genes. CDR HI, H2, and H3 loop residues as defined by Kabat scheme are identified by bold lines above. Sequence alignment was performed using Clustal-Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).
  • Figure 2B provides an amino acid sequence alignment of the parental VHH26 and six different humanized variants thereof.
  • CDR HI, H2, and H3 loop residues as defined by Kabat scheme are identified by bold lines above. Sequence alignment was performed using Clustal-Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).
  • Figures 3A-3B show biophysical characterization of the six humanized VHH26 variants (H1-H6).
  • Figure 3A shows SDS-PAGE of M-pull-down elution fractions from the metal-affinity chromatography.
  • SEC and Octect-BLI profiles of VHH26-H1, VHH26-H2, VHH26-H3, VHH26-H4, VHH26-H5, and VHH26-H6 humanized variants are summarized in the table of Figure 3B.
  • Figure 4 shows EC50 of binding to LRP5 or LRP6 of the indicated parental and variant VHH domains, in the context of the full engineered Wnt agonist format.
  • FIGS 5A-5D show in vitro activity of Fzd5,8 subfamily specific Wnt mimetic R2M13-26:
  • Figure 5A is a graph showing the binding affinity of the Fzd5,8 binder IgG of R2M13- 26 to its target Fzd5 CRD measured on Octet.
  • Figure 5B is a graph showing the binding affinity of the Fzd5,8 binder IgG of R2M13- 26 to its target Fzd8 CRD measured on Octet.
  • Figure 5C is a graph showing the binding specificity of the Fzd5,8 binder IgG of R2M13-26 to each of the 10 Fzd CRDs examined on Octet.
  • Figure 5D is a graph showing the dose-dependent STF activities of R2M13-26, of the Fzdl, 2, 7-specific mimetic 1RC07-26, and of the Fzdl,2,5,7,8 pan specific mimetic R2M3-26, in the presence of 20nM RSP02 measured in Huh-7 cells.
  • Figure 6 provides the sequences of the heavy chain and light chain present in the engineered WNT agonist, R2M13-h26.
  • FIG. 7 provides a schematic diagram of a DSS model of acute colitis and resulting serum antibody exposure following treatment with various non-humanized and humanized versions, including: R2M13-03-LALAPG (non-humanized), R2M13-26-LALAPG (non- humanized), R2M13-36-LALAPG (non-humanized), R2M13-humanized-03-LALAPG, R2M 13 -humanized-26-L ALAPG, R2M13-humanized-36-LAL APG, R2M 13 -humanized-03 - N297G, and R2M13-humanized-36-N297G.
  • R2M13-03-LALAPG non-humanized
  • R2M13-26-LALAPG non-humanized
  • R2M13-36-LALAPG non-humanized
  • R2M13-humanized-03-LALAPG R2M 13 -humanized-26-L ALAPG
  • Figure 8 provides graphs showing disease activity index of animals treated with the various non-humanized and humanized versions, including: R2M13-03-LALAPG (non- humanized), R2M13-26-LALAPG (non-humanized), R2M 13 -36-LAL APG (non-humanized), R2M 13 -humanized-03 -L ALAPG, R2M13-humanized-26-LAL APG, R2M 13 -humanized-36- L ALAPG, R2M13-humanized-03-N297G, and R2M 13 -humanized-36-N297G.
  • the lines of the graph from top to bottom correspond to: R2M13-h03-LALAPG, anti- GFP, R2M13-h03-N297G, R2M13-03-LALAPG, R2M13-36-LALAPG, R2M13-h36-N297G (behind R2M13 -h36-LAL APG), R2M13-h36-LALAPG, R2M13-h26-LALAPG, and no DSS, where “h” indicates humanized.
  • Figure 9 provides graphs showing levels of cytokines in animals treated with the various controls and non-humanized and humanized versions, including from left to right: no DSS, anti-GFP, parental R2M13-03-LALAPG (non-humanized), parental R2M13-26- L ALAPG (non-humanized), parental R2M13-36-LALAPG (non-humanized), R2M13- humanized-03-LALAPG, R2M13-humanized-26-LALAPG, R2M 13 -humanized-36- L ALAPG, R2M13 -humanized-03 -N297 G, and R2M13-humanized-36-N297G.
  • Figure 10 provides a graph showing levels of lipocalin 2 of animals treated with the various controls and non-humanized and humanized versions, including: no DSS, anti-GFP, parental R2M13-03-LALAPG (non-humanized), parental R2M13-26-LALAPG (non- humanized), parental R2M 13 -36-LAL APG (non-humanized), R2M 13 -humanized-03 - L ALAPG, R2M13 -humanized-26-LAL APG, R2M13 -humanized-36-LALAPG, R2M13- humanized-03-N297G, and R2M13-humanized-36-N297G.
  • Figure 11 provides micrographs showing restoration of epithelial tight junction marker, ZO-1, in vivo, in the DSS model of acute colitis, following treatment with the engineered WNT agonist. The brightly stained areas are ZO-1.
  • Figure 12 provides micrographs showing repair of damaged colon epithelium in vivo, in the DSS model of acute colitis, following treatment with the engineered WNT agonist, R2M13-h26-LALPG as compared to control anti-GFP.
  • Figure 13 provides micrographs showing restoration of the epithelial cell lineage including colonocytes, goblet cells, and tuft cells, in vivo, in the DSS model of acute colitis, following treatment with the engineered WNT agonist, R2M13-h26-LALPG as compared to control anti-GFP.
  • Figure 14 provides a graph and table showing pharmacokinetics (PK) of the parental R2M13-26-LALAPG and humanized R2M13-26-LALAPG following intravenous injection as determined by measuring the amount of antibody in serum at various times following administration to rats, and compared to data obtained from mice.
  • PK pharmacokinetics
  • Figure 15 provides a schematic diagram of an acute chronic colitis DSS animal model system.
  • Figure 16 provides graphs showing the disease activity index (DAI) of animals treated with R2M13-h26-LALAPG (R2M13-h26) or R2M13-26-LALAPG (R2M13-26).
  • DAI disease activity index
  • the lines of the graph from top to bottom correspond to: anti-GFP, cyclosporine A, R2M13-h26 (2 mpkxl), R2M13-h26 (20 mpkxl), R2M13-h26 (1 mpkx2), R2M13-h26 (6 mpkxl), R2M13-26 (3 mpkx2), R2M13-h26 (10 mpkx2), R2M13-26 (10 mpkx2), and no DSS.
  • Figure 17 shows a cross section of transverse colon with H&E staining of animals treated R2M13-h26, as compared to anti-GFP or cyclosporin A.
  • Figure 18 provides a diagram of a chronic DSS colitis animal model.
  • Figure 19 shows micrographs of transverse colon section following the indicated treatment.
  • Figure 20 provides graphs showing histology score and overall disease index following the indicated treatments.
  • Figure 21 provides graphs showing lipocalin-2 and IL-6 expression following the indicated treatments.
  • Figure 22 is a diagram of a chronic DSS colitis animal model.
  • Figure 23 provides graphs showing disease activity index of animals treated with
  • Figure 24 provides graphs showing expression of the indicated cytokines in animals treated with R2M13-h26 or IL12/23p40.
  • Figure 25 provides graphs showing Axin2 and Ki67 expression following the indicated treatments with R2M13-26-LALAPG (R2M13-26).
  • Figures 26A-26C shows the different cell types detected in the colon from scRNA- seq on uninjured and DSS-treated mice:
  • Figure 26A is a schematic diagram showing the experimental design of the scRNA seq experiment.
  • Figure 26B is a plot of the first two principal components: the lineage/tissue layer is indicated, showing the three groups radiating from the center.
  • Figure 26C provides graphs showing the strong impact the DSS injury had on number of differential genes expressed in different tissue layer/lineages.
  • the graph on the left shows the number of differentially expressed genes from each tissue layer on Day 5 and Day 6 of DDS mice compared to uninjured mice; the graph on the right shows the number of differentially expressed genes from each tissue layer on Day 5 and Day 6 of treatment with R2M13-26 compared to Anti-GFP.
  • the tissues/lineages from top to bottom of each bar correspond to epithelium, immune, and stroma, with almost all epithelium following treatment with R2M13-26-LALAPG (R2M13-26) at day 5.
  • FIGs 27A-27C shows that while DSS impacts all tissue layers by day 5, the predominant effect of R2M13-26-LALAPG (R2M13-26) is on the epithelium at 24-hours after treatment on day 5.
  • Figures 27A-27C showR2M13-26-LALAPG (R2M13-26) increased Wnt target and cell cycle gene expression and expanded the progenitors in the epithelium after injury.
  • Figure 27A is a table listing selected top gene sets (from GSEA) enriched in the R2M13-26 treated DSS-injured epithelium relative to the anti-GFP treated DSS-injured epithelium.
  • Figure 27B and 27C show validation of the scRNA-seq analysis in the tissue.
  • Figure 27B shows RNA in situ hybridization of two Wnt target genes, Axin2 and Cdkn3, in the uninjured, DSS/anti-GFP and DSS/ R2M13-26 treatment groups (day 5); nuclei labeled with DAPI. Scale bar represents 100 microns.
  • Figure 27C shows immunohistochemistry for the proliferative cell marker, MKI67, in the uninjured, DSS/anti-GFP, and DSS/ R2M13-26 treated colon samples (day 6); nuclei labeled with DAPI. Scale bar represents 100 microns.
  • FIGS 28A-28E show R2M13-26-LALAPG (R2M13-216) treatment caused accelerated, proper differentiation in the DSS model:
  • Figures 28A-D provide graphs showing uniform manifold approximation projection (UMAP) plots of the epithelial cells.
  • UMAP uniform manifold approximation projection
  • Figure 28A is a graph showing UMAP of epithelial cells colored by cluster/cell type.
  • Figure 28B is a graph showing UMAP colored by experimental condition of the cells.
  • Figure 28C is a graph showing a minimum spanning tree of the cluster medoids, connecting clusters based on similarity. Only the cell types that were not populated almost exclusively by injured cells were included. The stem cell and TA2 cell types were merged and set as the starting cluster.
  • Figure 28D is a graph showing the completed s7/ «gs/zo/-predicted lineage trajectory indicating a transition from the stem cell/TA cells to the EnteroPrecur cells on the way to the immature and mature enterocytes (going up); and bifurcating from the stem cell/TA cells down to go either toward tufted cells or toward goblet and enteroendocrine cells with a second bifurcation between them from the goblet progenitor cell type.
  • Figure 28E provides histograms of the number of cells from the indicated treatment groups at the 48-hour/day 6 timepoint at the indicated position along the pseudotime or lineage trajectory axis derived from the enterocyte lineage presented in Figure 28D.
  • the vertical red dashed line represents the same position along the axis in all three plots while the distribution shows how many cells are present at the position.
  • the pseudotime order (x-axis) is the same in each plot and is ordered from left to right.
  • Figure 28E shows that the progression toward enterocyte lineage is increased with R2M13-26- LALPG (R2M13-26) treatment.
  • Figures 29A-29L and 29A’-29L’ show that the Frizzled family of receptors presented differential expression patterns in the small intestinal epithelium:
  • Figures 29A-29L provide graphs showing expression of each of the 10 Fzd receptors (Fzdl-10), Axin2, and Lgr5, respectively, in the normal duodenum as determined by RNAscope in situ hybridization.
  • Figures 29A’-29’L provide graphs with zoomed in views showing Fzd expression in the small intestinal crypts. Arrows in panel E’ indicate intestinal stem cells.
  • Figures30A-30T show that the Frizzled family of receptors were expressed at different levels in the colon:
  • Figures 30A-30J provide graphs showing colon expression of the 10 Fzd receptors in naive mice examined by RNAscope in situ hybridization.
  • Figures 30K-30T provide graphs showing colon expression of the 10 Fzd receptors in mice treated with 7 days of 4% DSS.
  • Figure 31 shows a reduction of inflammation by a reduction of the neutrophil infiltrate.
  • S100A9 is a marker of neutrophil infiltration
  • CD45 is a marker of activated inflammatory cells.
  • Figure 32 provides a graph showing increased serum ALP following administration of the indicated dosages of R2M13-h26.
  • Figure 33 is a schematic drawing depicting a pharmacokinetic assay used to measure mean serum concentration of R2M13-h26.
  • Figure 34 provides a graph showing mean serum concentrations of R2M13-h26 in groups 2-4.
  • Figure 35 provides a graph showing individual serum R2M13-h26 concentrations measured following the first dose.
  • the arrow points to two animals in the 30 mg/kg dose group with accelerated clearance starting 3 days after dosing.
  • Figures 36A-36B provide a pair of graphs showing ALP increase in days 0-7 (Figure 36A) and days 28-42 (Figure 36B) for different dosage groups of R2M13-h26.
  • Figure 37 provides a graph showing mean serum R2M13-h26 concentrations after a single dose of R2M13-h26.
  • Figure 38 provides a table showing PK parameters for R2M13-h26 after a single dose of R2M13-h26.
  • “Activity” of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor, to catalytic activity, to the ability to stimulate gene expression, to antigenic activity, to the modulation of activities of other molecules, and the like. “Activity” of a molecule may also refer to activity in modulating or maintaining cell-to-cell interactions, e.g., adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton. “Activity” may also mean specific activity, e.g., [catalytic activity]/[mg protein], or [immunological activity ]/[mg protein], or the like
  • administering refers to delivery of a composition to a cell, to cells, to tissues, to tissue organoids, and/or to organs of a subject, or to a subject. Such administering or introducing may take place in vivo, in vitro or ex vivo.
  • an antibody means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binding the specific target antigen. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, VHH antibodies, camelid antibodies, nanobodies, diabodies, multi- specific antibodies (e.g., bispecific antibodies), and antibody fragments including but not limited to scFv, Fab, and Fab2, so long as they exhibit the desired biological activity.
  • Antibody fragments comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (e.g., Zapata et ah, Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (e.g, scFv); and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and 30 additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • a binding agent e.g., a Engineered WNT agonist or binding region thereof, or a WNT antagonist
  • a WNT antagonist is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • antigen-binding fragment refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chain, or of a Nanobody® (Nab), that binds to the antigen of interest, in particular to one or more FZD receptors, or to LRP5 and/or LRP6.
  • an antigen-binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL from antibodies that bind one or more FZD receptors or LRP5 and/or LRP6.
  • biological activity and “biologically active” refer to the activity attributed to a particular biological element in a cell.
  • biological activity of an WNT agonist, or fragment or variant thereof refers to the ability to mimic or enhance WNT signals.
  • biological activity of a polypeptide or functional fragment or variant thereof refers to the ability of the polypeptide or functional fragment or variant thereof to carry out its native functions of, e.g., binding, enzymatic activity, etc.
  • a functional fragment or variant retains at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of an activity of the corresponding native protein or nucleic acid.
  • the biological activity of a gene regulatory element e.g. promoter, enhancer, Kozak sequence, and the like, refers to the ability of the regulatory element or functional fragment or variant thereof to regulate, i.e. promote, enhance, or activate the translation of, respectively, the expression of the gene to which it is operably linked.
  • bifunctional antibody refers to an antibody that comprises a first arm having a specificity for one antigenic site and a second arm having a specificity for a different antigenic site, i.e., the bifunctional antibodies have a dual specificity.
  • Bispecific antibody is used herein to refer to a full-length antibody that is generated by quadroma technology (see Milstein et al., Nature, 305(5934): 537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see, Staerz et al., Nature, 314(6012): 628-631 (1985)), or by knob-into-hole or similar approaches, which introduce mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci USA, 90(14): 6444-6448 (1993)), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody.
  • a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC).
  • a bispecific antibody has two distinct antigen-binding arms (in both specificity and CDR sequences), and is monovalent for each antigen to which it binds.
  • an expression cassette “comprising” a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, e.g. poly-adenylation sequence, enhancer elements, other genes, linker domains, etc.
  • an expression cassette “consisting essentially of’ a gene encoding a therapeutic polypeptide operably linked to a promoter and a polyadenylation sequence may include additional sequences, e.g ., linker sequences, so long as they do not materially affect the transcription or translation of the gene.
  • a variant, or mutant, polypeptide fragment “consisting essentially of’ a recited sequence has the amino acid sequence of the recited sequence plus or minus about 10 amino acid residues at the boundaries of the sequence based upon the full length naive polypeptide from which it was derived, e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue less than the recited bounding amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the recited bounding amino acid residue.
  • control element or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter.
  • An “epitope” is specific region on an antigen that an antibody recognizes and binds to, and is also referred to as the “antigenic determinant”.
  • An epitope is usually 5-8 amino acids long on the surface of the protein. Proteins are three dimensionally folded structures, and an epitope may only be recognized in its form as it exists in solution, or its native form. When an epitope is made up of amino acids that are brought together by the three-dimensional structure, the epitope is conformational, or discontinuous. If the epitope exists on a single polypeptide chain, it is a continuous, or linear epitope. Depending on the epitope an antibody recognizes, it may bind only fragments or denatured segments of a protein, or it may also be able to bind the native protein.
  • the portion of an antibody or antibody fragment thereof that recognizes an epitope is referred to as the “epitope binding domain” or “antigen binding domain”.
  • the epitope or antigen binding domain of an antibody or antibody fragment is in the Fab fragment and the effector functions in the Fc fragment.
  • Six segments, known as complementarity determining regions (CDRs) within the variable regions (VH and VL) of the heavy and light chains loop out from the framework (FR regions) globular structure of the rest of the antibody and interact to form an exposed surface at one end of the molecule. This is the antigen binding domain.
  • CDRs complementarity determining regions
  • VH and VL variable regions
  • FR regions framework regions
  • An "expression vector” is a vector, e.g. plasmid, minicircle, viral vector, liposome, and the like as discussed herein or as known in the art, comprising a region which encodes a gene product of interest, and is used for effecting the expression of the gene product in an intended target cell.
  • An expression vector also comprises control elements, e.g., promoters, enhancers, UTRs, miRNA targeting sequences, etc., operatively linked to the encoding region to facilitate expression of the gene product in the target.
  • control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.
  • FR set refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface.
  • Humanized antibodies or fragments thereof refers to antibodies or fragments thereof from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. The process of "humanization” is usually applied to monoclonal antibodies developed for administration to humans.
  • the terms "individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).
  • mammalian sport animals e.g., horses
  • mammalian farm animals e.g., sheep, goats, etc.
  • mammalian pets dogs, cats, etc.
  • rodents e.g., mice, rats, etc.
  • a “monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope.
  • monoclonal antibody encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab 1 , F(ab')2, Fv), single chain (scFv), Nanobodies®, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen- binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope, including Engineered WNT agonists disclosed herein.
  • fragments thereof such as Fab, Fab 1 , F(ab')2, Fv), single chain (scFv), Nanobodies®, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immuno
  • antibody it is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).
  • the term includes whole immunoglobulins as well as the fragments etc. described herein or under the definition of "antibody”.
  • mutant refers to a mutant of a reference polynucleotide or polypeptide sequence, for example a native polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence.
  • a variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polynucleotide sequence, e.g., a native polynucleotide or polypeptide sequence.
  • a variant may be a polynucleotide having a sequence identity of 50% or more, 60% or more, or 70% or more with a full-length native polynucleotide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native polynucleotide sequence.
  • a variant may be a polypeptide having a sequence identity of 70% or more with a full-length native polypeptide sequence, e.g. , an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native polypeptide sequence.
  • Variants may also include variant fragments of a reference, e.g., native, sequence sharing a sequence identity of 70% or more with a fragment of the reference, e.g., native, sequence, e.g. , an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the native sequence.
  • “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” refer to polymers of amino acids of any length The terms also encompass an amino acid polymer that has been modified; for example, to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences.
  • identity and “identical” refer, with respect to a polypeptide or polynucleotide sequence-of-interest, to the percentage of exact matching residues in an alignment of that the sequence-of-interest to a reference sequence, such as an alignment generated by the BLAST algorithm. Identity is calculated, unless specified otherwise, across the full length of the reference sequence.
  • a sequence-of-interest “shares at least x% identity to” a reference sequence if, when the reference sequence is aligned (as a query sequence) is aligned to the sequence-of-interest (as subject sequence), at least x% (rounded down) of the residues in the subject sequence are aligned as an exact match to a corresponding residue in the query sequence, the denominator being the full length of the reference sequence plus the lengths of any gaps inserted into the reference sequence by alignment of the reference sequence to the sequence-of-interest.
  • the subject sequence has variable positions (e.g., residues denoted X), an alignment to any residue in the query sequence is counted as a match.
  • Sequence similarity can be determined in a number of different manners
  • sequences can be aligned using the methods and computer programs, including BLAST, available over the worldwide web at ncbi.nlm nih.gov/BLAST/. Sequence alignments may be performed using the NCBI Blast service (BLAST+ version 2.12.0) or another program giving the same results. Unless indicated to the contrary, sequence identity is determined using the BLAST algorithm (e.g., bl2seq) with default parameters.
  • FASTA Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis , USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co , San Diego, Calif., USA.
  • alignment programs that permit gaps in the sequence.
  • the Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997)
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970)
  • the program has default parameters determined by the sequences inputted to be compared.
  • the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA.
  • GCG Genetics Computing Group
  • FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.
  • a "promoter” as used herein encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues and species or cell-type specific, tissue-specific, or species specific. Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Also included in the nucleic acid constmcts or vectors of the invention are enhancer sequences that may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5' or 3' regions of the native gene.
  • Recombinant, as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • treatment used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, e.g., reducing the likelihood that the disease or symptom thereof occurs in the subject, and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during or after the onset of disease or injury.
  • the treatment of ongoing disease where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues.
  • the subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e , the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • the present invention provides compositions and methods of modulating WNT signals to ameliorate various diseases and disorders that may benefit from modulation of WNT signaling pathways, such as gastrointestinal disorders, including but not limited to, inflammatory bowel disease, including but not limited to, Crohn’s disease, Crohn’s disease with fistula formation, and ulcerative colitis.
  • gastrointestinal disorders including but not limited to, inflammatory bowel disease, including but not limited to, Crohn’s disease, Crohn’s disease with fistula formation, and ulcerative colitis.
  • WNT Wired-related integration site
  • WNT Wired and Int-1
  • Wingless- Int-1 Wingless- Int
  • ligands and their signals play key roles in the control of development, homeostasis and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste bud, ovary, cochlea, lung, and many other tissues (reviewed, e.g., by Clevers, Loh, and Nusse, 2014; 346:1248012). Modulation of WNT signaling pathways has potential for treatment of degenerative diseases and tissue injuries.
  • LRP5 and LRP6 are collectively referred to as LRP5/6, and reference to “LRP5/6 binding,” or the like, indicates binding to LRP5 and/or LRP6.
  • R-spondins 1-4 are a family of ligands that amplify WNT signals. Each of the R-spondins works through a receptor complex that contains Zinc and Ring Finger 3 (ZNRF3) or Ring Finger Protein 43 (RNF43) on one end and a Leucine-rich repeat-containing G-protein coupled receptor 4-6 (LGR4-6) on the other (reviewed, e.g., by Knight and Hankenson 2014, Matrix Biology; 37: 157-161). R-spondins might also work through additional mechanisms of action.
  • ZNRF3 and RNF43 are two membrane-bound E3 ligases specifically targeting WNT receptors (FZDl-10 and LRP5 or LRP6) for degradation.
  • R-spondin binding to ZNRF3/RNF43 and LGR4-6 causes clearance or sequestration of the ternary complex, which removes E3 ligases from WNT receptors and stabilizes WNT receptors, resulting in enhanced WNT signals.
  • Each R-spondin contains two Furin domains (1 and 2), with Furin domain 1 binding to ZNRF3/RNF43, and Furin domain 2 binding to LGR4-6. Fragments of R-spondins containing Furin domains 1 and 2 are sufficient for amplifying WNT signaling. While R-spondin effects depend on WNT signals, since both LGR4-6 and ZNRF3/RNF43 are widely expressed in various tissues, the effects of R-spondins are not tissue-specific.
  • Activating WNT signaling by a WNT agonist may be used for the treatment of a variety of diseases and disorders, including gastrointestinal disorders.
  • amplifying WNT signaling by RSPO or an RSPO mimetic may be used for the treatment of a variety of diseases and disorders, including gastrointestinal disorders.
  • RSPO may be used for the treatment of experimental colon colitis (J. Zhao et. al., 2007).
  • a WNT agonist molecule may also be used for the treatment of gastrointestinal disorders.
  • active WNT signaling can provide a major stem cell maintenance signal and plays a key role in regulating regeneration of the intestinal epithelium in homeostasis and in injury.
  • Secretory cells secrete hormones and provide an important barrier against food-borne microorganisms, toxins, and antigens, mainly through the secretion of mucus and anti-microbial peptides.
  • the absorptive cells conduct uptake of dietary nutrients, as they localize mainly at the tips of the villi in the small intestine or at the top of the colonic crypts, thus constituting the majority of luminal cells across the intestinal surface area (see, e.g., Santos, et. al (2016) Trends in Cell Biol. in press, https://doi.Org/10.1016/j.tcb.2018.08.001). Under homeostasis conditions, all cells in the intestinal epithelium regenerate in 3-10 days.
  • ISC intestinal stem cell
  • BMP Bone Morpohogenetic Protein
  • This regeneration is mediated by either surviving Lgr5+ ISCs or other mature cell types such as enterocytes, enteroendocrine, or Paneth cells that can convert back to Lgr5+ ISCs to aid epithelial regeneration (Beumer and Clevers (2016), Development 143: 3639-3649).
  • ISCs at the bottom of the intestinal crypt also known as columnar base cells (CBCs)
  • CBCs columnar base cells
  • WNT secreting Paneth cells Chole and Leblond (1974) Am. J. Anat. 141: 537-561).
  • Mesenchymal cells surrounding the intestinal epithelium also secrete some WNT proteins, serving an overlapping stem cell niche function in vivo (Farin, el. al (2012) Gastroenterol. 143: 1518-1529).
  • WNT signaling ISCs divide to produce self-renewing stem cells and differentiating daughter cells, which first go through a few fast transit amplifying (TA) divisions before differentiating into functional cell types.
  • TA fast transit amplifying
  • the present disclosure provides engineered WNT agonists and contemplates the use of engineered WNT agonists to stimulate, agonize, or promote WNT signaling, e.g., through the canonical WNT/p-catenin signaling pathway.
  • engineered WNT agonists may also be referred to as WNT/b -catenin signaling agonists or Wnt mimetics.
  • Wnt proteins are difficult to produce and do not contain typical drug-like properties.
  • the disclosure addresses the first challenge by providing synthetic Wnt mimetics with drug-like properties, particularly in the form of recombinant, bi-specific antibodies that bring together Fzd and Lrp to stimulate signaling, mimicking endogenous Wnt ligands.
  • the Wnt mimetics of the disclosure may freely diffuse, access damaged tissues and guide tissue repair where Wnt signals are needed.
  • the disclosure addresses the second challenge by providing Wnt mimetics that are capable of repairing damaged intestine epithelium without being combined with RSPO. Unlike RSPO, the Wnt mimetics of the disclosure do not induce hyperproliferation of normal intestine epithelium. [00117] Wnt mimetics of the disclosure have the desired properties of restoring diseased intestine tissue back to normal physiology. In some embodiments, Wnt mimetics of the disclosure induce rapid restoration of damaged epithelial tissue. In some embodiments, damaged epithelial barrier may be restored within about 10 days, about 8 days, about 7 days, about 6 days, or about 5 days of treatment with Wnt mimetics of the disclosure.
  • damaged epithelial barrier may be restored within about 6 days of treatment with Wnt mimetics of the disclosure.
  • Wnt mimetics of the disclosure induce expression of Wnt target genes in injured epithelial cells within about 12 hours, about 24 hours, about 36 hours, or about 48 hours.
  • Wnt mimetics of the disclosure induce expression of Wnt target genes in injured epithelial cells within about 24 hours.
  • the Wnt target genes that are induced by Wnt mimetics of the disclosure comprise Axin2, Rnf43, Cdkn3.
  • expression of Axin2 in injured epithelial cells is induced by Wnt mimetics of the disclosure within about 24 hours.
  • the Wnt agonists disclosed herein support the proliferation and differentiation of stem cells in the damaged intestinal or colonic crypts of patients with moderate to severe IBD.
  • the Wnt agonists disclosed herein have the potential to accelerate the repair of the intestinal barrier, which can result in a reduction of bacteria penetrating through the intestinal epithelium and a reduction of immune cell activation and inflammation, thereby treating inflammatory bowel diseases.
  • the Wnt agonists disclosed herein have several simultaneous beneficial effects: activate the Wnt signaling pathway in intestinal stem cells and progenitor cells resulting in proliferation and differentiation; restore intestinal barrier function and tissue architecture; reduce tissue inflammation; and reduce disease activity in moderate to severe IBD.
  • the Wnt agonists disclosed herein is a bispecific antibody targeting Fzd5/8 and Lrp6.
  • Fzd5 was previously reported to be highly expressed in intestinal mucosal cells from IBD patients.
  • Fzd5 was also highly expressed in a mouse model of colitis induced by dextran sodium sulfate (DSS).
  • DSS dextran sodium sulfate
  • the Wnt agonists disclosed herein binds to DSS-injured intestinal cells, stimulating Wnt signaling as measured by the expression of Axin2, a downstream target gene in the Wnt pathway.
  • the Wnt agonists disclosed herein binds to Fzd5/8 and Lrp6 on intestinal stem cells to activate Wnt signaling.
  • administration of a Wnt agonist disclosed herein improves in the disease activity index, or DAI, in a DSS model.
  • the DAI is a composite score composed of body weight change, diarrhea, and bloody stools that is frequently used to quantify disease severity in preclinical rodent models and in the clinic.
  • administration of a Wnt agonist disclosed herein leads to a dose dependent decrease in DAI.
  • treatment with a Wnt agonist disclosed herein is superior to treatment with cyclosporine, an anti-TNF antibody, or an anti-IL12/23 antibody.
  • administration of a Wnt agonist disclosed herein improves the DAI in both a chronic DSS model and an acute DSS model.
  • Wnt mimetics of the disclosure expand progenitor cell populations in the epithelium.
  • Wnt mimetics of the disclosure expand progenitor cell populations by increasing the expression of cell cycle genes in said cell populations.
  • the progenitor cell populations may include, for example, normal progenitors responding to injury and progenitors in altered cell states, such as de-differentiation.
  • Wnt mimetics of the disclosure substantially expand progenitor cell populations in the epithelium within about 24 hours.
  • Wnt mimetics of the disclosure accelerates differentiation of progenitor cells into mature cell types.
  • Wnt mimetics of the disclosure accelerates differentiation of progenitor cells, e.g., gastrointestinal progenitor cells, to enterocytes, goblet cells, enteroendocrine, or tuft cells.
  • Wnt mimetics of the disclosure accelerate differentiation of progenitor cells to enterocytes.
  • substantial differentiation of progenitor cells into mature cell types occurs within about 24 hours, about 36 hours, about 48 hours, or about 60 hours of treatment with Wnt mimetics of the disclosure.
  • substantial differentiation of progenitor cells into mature cell types occurs within about 48 hours of treatment with Wnt mimetics of the disclosure.
  • Wnt mimetics of the disclosure accelerate differentiation of progenitor cells into mature cell types while reducing expression of high levels of inflammatory genes
  • the breakdown of the intestinal barrier triggers influx of luminal pathogen and an inflammatory response that leads to further tissue damage.
  • Disease modification in IBD can be measured by the levels of inflammatory cytokines present in the injured tissue and in serum.
  • treatment of epithelial tissue injury with Wnt mimetics of the disclosure reduces production of inflammatory cytokines.
  • treatment of damaged epithelial tissue with Wnt mimetics of the disclosure reduces inflammatory cytokine production by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30% at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to a treatment that does not comprise Wnt mimetics of the disclosure , or compared to no treatment.
  • the disclosure provides a Wnt mimetic capable of effectively repairing injured epithelium without inducing over proliferation of normal epithelium. In some embodiments, Wnt mimetics of the disclosure alone does not affect proliferation of normal epithelium. In some embodiments, the epithelium is colon or small intestine epithelium. In some embodiments, the disclosure provides a Wnt mimetic capable of repairing injured epithelium with higher efficacy than a treatment comprising RSPO. In some embodiments, the disclosure provides a Wnt mimetic capable of repairing injured epithelium with higher efficacy than a treatment comprising RSPO and a Wnt mimetic of the disclosure.
  • the Wnt mimetic of the disclosure improves injured epithelium with better efficacy than a treatment comprising RSPO, or a treatment comprising RSPO and a Wnt mimetic of the disclosure, by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30% at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% Efficacy of repairing injured epithelium may be determined by histology severity scores wherein a higher score indicates more severe damage, or by disease activity index (DIA), which may be calculated based on the average score of weight loss, stool consistency and the degree of intestinal bleeding.
  • DIA disease activity index
  • the disclosure provides a Fzd5,8 and Lrp6-specific Wnt mimetic- (for example, R2M13-26 or R2M13-h26).
  • the Fzd5,8 and Lrp6-specific Wnt mimetic of the disclosure is capable of activating Wnt signaling on epithelial cells Activation of Wnt signaling may be measured by gene expression using scRNA-seq (single-cell RNA sequencing) methods known in the art and described in the disclosure.
  • the epithelium cells are colon or small intestine epithelium cells.
  • the epithelial cells include multiple stem or progenitor cells.
  • the engineered WNT agonists include one or more binding domain that binds to one or more FZD or an epitope thereof, and one or more binding domain that binds to one or more of LRP5 and/or LRP6, or an epitope within LRP5 and/or LRP6.
  • the engineered WNT agonist specifically binds to the cysteine-rich domain (CRD) within the human frizzled receptor(s) to which it binds.
  • the engineered WNT agonists may comprise one or more additional binding domain.
  • they may comprise one or more binding domains that bind to one or more of the E3 ligases, ZNRF3/RNF43, or specific epitopes within either of the E3 ligases.
  • the E3 ligase binding domain comprises an R-SPO or a fragment thereof.
  • the engineered WNT agonists may comprises one or more tissue-specific or cell type-specific binding domain that specifically binds to a target tissue or cell type.
  • the disclosure provides VHH domains that bind to LRP5 and/or LRP6. Illustrative sequences of these VHH domains are provided in Table 1.
  • the VHH binding domains may be derived from any of the disclosed sequences.
  • the VHH binding domains are humanized.
  • the present disclosure contemplates engineered WNT agonists that comprise one of more disclosed VHH domain, including any of the humanized VHH domains disclosed herein, as well functional fragments and variants of such VHH domains having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the VHH sequences disclosed herein.
  • a VHH domain comprises three CDR sequences: GREFAIYDIA, IRPVVTEIDYADSVKG, and RPWGSRDEY.
  • an engineered Wnt agonist comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 19-25.
  • the engineered Wnt agonist is a bi-specific antibody-like molecule comprising an IgG structure comprising two heavy chains and two light chains, wherein VHH domains are fused to the N-terminus of each light chain present in the antibody-like molecule.
  • the heavy chain is effector-less, e.g., contains LALAPG mutations.
  • the disclosure provides FZD binding domains that bind to one or more FZD.
  • Illustrative sequences of these FZD binding domain are provided in Table 3, in the context of VH and VL domains derived from an anti -FZD antibody, R2M13.
  • the present disclosure contemplates engineered WNT agonists that comprise one or more of the VH or VL domains disclosed herein, as well functional fragments and variants of such VH or VL domains having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the VH or VL sequences disclosed herein.
  • the present disclosure contemplates engineered WNT agonists that comprise one or more of the heavy or light chain sequences provided in Table 3, as well functional fragments and variants of such heavy or light chains having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the heavy or light chain sequences disclosed herein.
  • a FZD binding domain comprises three light chain CDR sequences: RASQSISSYLN (CDRL1), AASSLQS (CDRL2), and QQSYSTPLT (CDRL3), and/or three heavy chain light chain CDR sequences: GGTFTYRYLH (CDRH1), GIIPIF GTGNYAQKFQG (CDRH2), and SMVRYPYYY GMD V (CDRH3), any CDRs provided herein.
  • the disclosure contemplates engineered WNT agonists comprising one or more CDRs present in a FZD binding domain or LRP5/6 binding domain disclosed herein: e.g., one or more (e.g, two or three) of the VHH CDRs shown in Figure 6 or Table 1; one or more (e.g., two or three) of the CDRs present in a heavy chain or light chain disclosed herein.
  • the engineered WNT agonists comprise 4, 5, or all six of the CDRs shown for a FZD binding domain disclosed herein, e.g., in Figure 6 or Table 3.
  • the engineered WNT agonists comprises 6, 7, 8, or all 9 of the CDRs shown for an engineered WNT agonists disclosed herein e.g., in Figure 6 or Table 3.
  • the disclosure provides polypeptides comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a binding domain provided herein, as well as polypeptides comprising two or more, e.g., three, of the CDR sequences disclosed herein, such as a polypeptide comprising the following CDRs: GRIFAIYDIA, IRPVVTEIDYADSVKG, and RPWGSRDEY (VHH CDRs 1-3, respectively), and which binds to LRP5 or LRP6, or a polypeptide comprising the following CDRs: RASQSISSYLN (CDRLl), AASSLQS (CDRL2), and QQSYSTPLT (CDRL3), which,
  • the disclosure also includes a FZD binding domain comprising two heavy chains and two light chains, wherein each heavy chain comprises two or more of the following CDRs: GGTFTYRYLH (CDRHl), GIIPIFGTGNYAQKFQG (CDRH2), and SMVRVPYYYGMDV (CDRH3), and each light chain comprises two or more of the following CDRs: RASQSISSYLN (CDRLl), AASSLQS (CDRL2), and QQSYSTPLT (CDRL3), wherein the FZD binding domain binds to one or more FZD.
  • CDRHl GGTFTYRYLH
  • CDRH2 GIIPIFGTGNYAQKFQG
  • CDRH3 SMVRVPYYYGMDV
  • CDRLl RASQSISSYLN
  • AASSLQS CDRL2
  • QQSYSTPLT CDRL3
  • the FZD binding domain is an antibody
  • the heavy chain further comprises an Fc domain, e.g., an IgGl Fc domain, which may be modified.
  • the disclosure further provides polypeptides comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a variable heavy or variable light domain disclosed herein e.g., in SEQ ID NOs: 1-25, Figure 6 or Table 3.
  • the disclosure further provides polypeptides comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a VHH domain disclosed herein e g., in SEQ DI NOs: 1-25, Figure 6 or Table 3.
  • the disclosure further provides polypeptides comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a heavy chain or light chain or fusion polypeptide disclosed herein e g., in SEQ ID NOs: 1-25, Figure 6 or Table 3.
  • the CDRs are not modified as compared to the original or parental sequence.
  • an engineered WNT agonist disclosed herein comprises an Fc domain (e.g., as part of a heavy chain).
  • the Fc domain is engineered to include specific amino acid substitutions, including those corresponding to LALAPG or N297G.
  • one or more LRP5/6 binding domain disclosed herein is fused to one or more of the light chain or heavy chain of a FZD binding domain disclosed herein (e.g., an R2M13 derived FZD binding domain), e.g., directly or via a linker, e.g., a peptide linker.
  • any LRP5/6 binding domain disclosed herein may be fused to or complexed with a different FZD binding domain to achieve an engineered WNT agonist
  • any FZD binding domain disclosed herein may be fused to or complexed with a different LRP5/6 binding domain to achieve an engineered WNT agonist.
  • a variety of anti-FZD or anti-LRP antibodies that may be present in whole or in part in an engineered WNT agonist disclosed herein include those described in U.S. Pat. No. 7,462,697, PCT Publication No. WO 2019/126399 , and PCT Publication No. WO 2019/126401. Illustrative formats and sequences are also provided in PCT Publication No. WO 2019/126398, each of which is incorporated herein in its entirety.
  • Engineered WNT agonists may adopt a variety of different structural conformations, each comprising one or more, e.g., two, FZD binding domains and one or more, e.g., two) LRP5/6 binding domains.
  • the FZD binding domain(s) and LRP5/6 binding domain(s) may be directly fused to each other or via a linker, e.g., a peptide linker.
  • the FZD binding domain(s) and LRP5/6 binding domain(s) may be complexed to each other.
  • the engineered WNT agonist comprises two heavy chains and two light chains, wherein the light chain comprises a fused VHH, and adopts an antibodylike confirmation, wherein the two heavy chains are bound to each other via disulfide bonds and the two light chains are bound to the heavy chains via disulfide bonds.
  • the engineered WNT agonists may adopt other antibody-like structures or confirmations, including those found in various functional fragments, including but not limited to any of those disclosed herein.
  • an antibody is an immunoglobulin molecule capable of specific binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least on epitope binding domain, located on the variable region of the immunoglobulin molecule.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof containing epitope binding domains (e.g ., dAb, Fab, Fab’, (F(ab’)2, Fv, single chain (scFv), camelid antibodies, Nanobodies® (Nabs; also known as sdAbs or VHH domains), DVD-Igs, synthetic variants thereof, naturally occurring variants, fusion proteins comprising and epitope binding domain, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity.
  • epitope binding domains e.g ., dAb, Fab, Fab’, (F(ab’)2, Fv, single chain (scFv), camelid antibodies, Nanobodies® (Nabs; also known as sdAbs or VHH domains), DVD-Igs, synthetic variants thereof, naturally occurring
  • Diabodies multivalent or multispecific fragments constmcted by gene fusion (WO94/13804, P. Holliger et ah, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) are also a particular form of antibody contemplated herein.
  • Minibodies comprising a scFv joined to a CH3 domain are also included herein (S. Hu et ah, Cancer Res., 56, 3055-3061, 1996). See e.g., Ward, E. S.
  • the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site.
  • the enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab')2 fragment which comprises both antigenbinding sites.
  • An Fv fragment for use according to certain embodiments of the present disclosure can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art.
  • the Fv fragment includes a non-covalent VH::VU heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule.
  • VH::VU heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule.
  • antibodies and antigen-binding fragments thereof as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other.
  • CDR set refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as "CDR1," "CDR2,” and “CDR3" respectively.
  • An antigen-binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • a polypeptide comprising a single CDR (e.g ., a CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit.” Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site. [00143] As used herein, the term "FR set" refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region.
  • FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain "canonical" structures — regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.
  • a “monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope.
  • monoclonal antibody encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab 1 , F(ab')2, Fv), single chain (scFv), Nanobodies®, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen- binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope, including Engineered WNT agonists disclosed herein.
  • fragments thereof such as Fab, Fab 1 , F(ab')2, Fv), single chain (scFv), Nanobodies®, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immuno
  • antibody it is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g ., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).
  • the term includes whole immunoglobulins as well as the fragments etc. described above under the definition of "antibody”.
  • single chain Fv or scFV antibodies are contemplated for use in the engineered Wnt agonists.
  • Kappa bodies Ill et al., Prot. Eng. 10: 949- 57 (1997)); minibodies (Martin et al., EMBO J 13: 5305-9 (1994)); diabodies (Holliger et al., PNAS 90: 6444-8 (1993)); or Janusins (Traunecker et al., EMBO J 10: 3655-59 (1991) and Traunecker et al., Int. J Cancer Suppl.
  • bispecific or chimeric antibodies may be made that encompass the ligands of the present disclosure.
  • a chimeric antibody may comprise CDRs and framework regions from different antibodies, while bispecific antibodies may be generated that bind specifically to one or more FZD receptors through one binding domain and to a second molecule through a second binding domain.
  • These antibodies may be produced through recombinant molecular biological techniques or may be physically conjugated together.
  • a single chain Fv (scFv) polypeptide is a covalently linked VH::VL heterodimer which is expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker.
  • a number of methods have been described to discern chemical structures for converting the naturally aggregated — but chemically separated — light and heavy polypeptide chains from an antibody V region into an scFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U S. Patent Nos.
  • an antibody as described herein is in the form of a diabody.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • a dAb fragment of an antibody consists of a VH domain (Ward, E. S. et ah, Nature 341, 544-546 (1989)).
  • bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G., Current Opinion Biotechnol. 4, 446-449 (1993)), e.g., prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
  • Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti -idiotypic reaction.
  • Bispecific diabodies as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by knobs-into-holes engineering (J. B. B. Ridgeway et ah, Protein Eng , 9, 616-621 (1996)).
  • the antibodies described herein may be provided in the form of a UniBody®.
  • a UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This proprietary antibody technology creates a stable, smaller antibody format with an anticipated longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and thus do not interact with the immune system. Fully human IgG4 antibodies may be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments having distinct stability properties relative to the corresponding intact IgG4 (GenMab, Utrecht).
  • the antibodies of the present disclosure may take the form of a single domain (sdAb) or VHH antibody fragment (also known as a Nanobody®).
  • sdAb single domain
  • VHH VHH antibody fragment
  • the sdAb or VHH technology was originally developed following the discovery and identification that camelidae (e.g., camels and llamas) possess fully functional antibodies that consist of heavy chains only and therefore lack light chains.
  • camelidae e.g., camels and llamas
  • These heavy-chain only antibodies contain a single variable domain(VHH) and two constant domains (CH2, CH3).
  • the cloned and isolated single variable domains have full antigen binding capacity and are very stable. These single variable domains, with their unique structural and functional properties, form the basis of “Nanobodies®”.
  • the sdAbs or VHHs are encoded by single genes and are efficiently produced in almost all prokaryotic and eukaryotic hosts, e.g., E. coli (see, e.g., U.S. Pat. No. 6,765,087), molds (for example Aspergillus or Trichoderma) and yeast (for example Saccharomyces, Kluyvermyces, Hansenula or Pichia (see, e.g., U.S. Pat. No. 6,838,254).
  • Nanobodies® The production process is scalable and multi-kilogram quantities of Nanobodies® have been produced.
  • sdAbs or VHHs may be formulated as a ready-to-use solution having a long shelf life.
  • the Nanoclone® method (see, e.g., WO 06/079372) is a proprietary method for generating Nanobodies® against a desired target, based on automated high-throughput selection of ficelle sdAbs or VHHs are single-domain antigen-binding fragments of camelid-specific heavy- chain only antibodies.
  • DVD-Ig dual-variable domain- immunoglobulin
  • a DVD-Ig is an engineered protein that combines the function and specificity of two monoclonal antibodies in one molecular entity.
  • a DVD-Ig is designed as an IgG-like molecule, except that each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage, instead of one variable domain in IgG.
  • the fusion orientation of the two variable domains and the choice of linker sequence are critical to functional activity and efficient expression of the molecule.
  • a DVD-Ig can be produced by conventional mammalian expression systems as a single species for manufacturing and purification.
  • a DVD-Ig has the specificity of the parental antibodies, is stable in vivo, and exhibits IgG-like physicochemical and pharmacokinetic properties. DVD-Igs and methods for making them are described in Wu, C., et ab, Nature Biotechnology, 25:1290-1297 (2007)).
  • the antibodies or antigen-binding fragments thereof as disclosed herein are humanized. This refers to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin.
  • the antigen-binding site may comprise either complete variable domains fused onto constant domains or only the CDRs grafted onto appropriate framework regions in the variable domains.
  • Epitope binding sites may be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A. F. et ab, (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327).
  • variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs.
  • CDRs complementarity-determining regions
  • FRs framework regions
  • humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies).
  • humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody.
  • the antibodies of the present disclosure may be chimeric antibodies.
  • a chimeric antibody is comprised of an antigen-binding fragment of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody
  • the heterologous Fc domain is of human origin.
  • the heterologous Fc domain may be from a different Ig class from the parent antibody, including IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3, and IgG4), and IgM.
  • the heterologous Fc domain may be comprised of CH2 and CH3 domains from one or more of the different Ig classes.
  • the antigen-binding fragment of a chimeric antibody may comprise only one or more of the CDRs of the antibodies described herein (e.g ., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or may comprise an entire variable domain (VL, VH or both).
  • immunoglobulin CDRs and variable domains may be determined by reference to Rabat, E. A. et ah, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (immuno.bme.nwu edu).
  • Engineered WNT agonist comprises one or more Fab or antigen-binding fragment thereof and one or more VHH or sdAb or antigen-binding fragment thereof (or alternatively, one or more scFv or antigen-binding fragment thereof).
  • the Fab specifically binds one or more Fzd receptor
  • the VHH or sdAb (or scFv) specifically binds LRP5 and/or LRP6.
  • the Fab specifically binds LRP5 and/or LRP6, and the VHH or sdAb (or scFv) specifically binds one or more Fzd receptor.
  • the VHH or sdAb (or scFv) is fused to the N-terminus of the Fab, while in some embodiments, the VHH or sdAb (or scFv) is fused to the C-terminus of the Fab.
  • the Fab is present in a full IgG format, and the VHH or sdAb (or scFv) is fused to the N-terminus and/or C-terminus of the IgG light chain.
  • the Fab is present in a full IgG format, and the VHH or sdAb (or scFv) is fused to the N-terminus and/or C-terminus of the IgG heavy chain.
  • two or more VHH or sdAb are fused to the IgG at any combination of these locations.
  • Fabs may be converted into a full IgG format that includes both the Fab and Fc fragments, for example, using genetic engineering to generate a fusion polypeptide comprising the Fab fused to an Fc region, i.e., the Fab is present in a full IgG format.
  • the Fc region for the full IgG format may be derived from any of a variety of different Fes, including but not limited to, a wild-type or modified IgGl, IgG2, IgG3, IgG4 or other isotype, e.g., wild-type or modified human IgGl, human IgG2, human IgG3, human IgG4, human IgG4Pro (comprising a mutation in core hinge region that prevents the formation of IgG4 half molecules), human IgA, human IgE, human IgM, or the modified IgGl referred to as IgGl LALAPG.
  • the L235 A, P329G (LALA-PG) variant has been shown to eliminate complement binding and fixation as well as Fc-g dependent antibody-dependent cell-mediated cytotoxity (ADCC) in both murine IgG2a and human IgGl.
  • ADCC Fc-g dependent antibody-dependent cell-mediated cytotoxity
  • the IgG comprises one or more of the following amino acid substitutions: N297G, N297A, N297E, L234A, L235A, or P236G.
  • Non-limiting examples of bivalent and bispecific Engineered WNT agonists that are bivalent towards both the one or more Fzd receptor and the LRP5 and/or LRP6 are provided, inclduing but not limited to those provided in Table 3
  • the VHH or sdAb (or scFvs) may be fused to the N-termini of both light chains, to the N-termini of both heavy chains, to the C-termini of both light chains, or to the C-termini of both heavy chains.
  • VHH or sdAb could be fused to both the N-termini and C- termini of the heavy and/or light chains, to the N-termini of the light chains and the heavy chains, to the C-termini of the heavy and light chains, to the N-termini of the heavy chains and C-termini of the light chains, or to the C-termini of the heavy chains and the N-termini of the light chains.
  • two or more VHH or sdAb (or scFvs) may be fused together, optionally via a linker moiety, and fused to the Fab or IgG at one or more of these locations.
  • the Engineered WNT agonist has a Hetero-IgG format, whereas the Fab is present as a half antibody, and one or more VHH or sdAb (or scFv) is fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, the C-terminus of the Fc, or the C-terminus of the Fab.
  • the Fab or antigen-binding fragment (or IgG) thereof is fused directly to the VHH or sdAb (or scFv) or antigen-binding fragment thereof, whereas in other embodiments, the binding regions are fused via a linker moiety.
  • a Engineered WNT agonist comprises one or more Fab or antigen-binding fragment thereof that binds one or more FZD receptor and one or more Fab or antigen-binding fragment thereof that binds LRP5 and/or LRP6. In certain embodiments, it comprises two Fab or antigen-binding fragments thereof that bind one or more FZD receptor and/or twoFab or antigen-binding fragments thereof that bind LRP5 and/or LRP6. In particular embodiments, one or more of the Fab is present in a full IgG format, and in certain embodiments, both Fab are present in a full IgG format.
  • the Fab in full IgG format specifically binds one or more FZD receptor, and the other Fab specifically binds LRP5 and/or LRP6. In certain embodiments, the Fab specifically binds one or more FZD receptor, and the Fab in full IgG format specifically binds LRP5 and/or LRP6. In certain embodiments, the Fab specifically binds LRP5 and/or LRP6, and the Fab in full IgG format specifically binds one or more FZD receptor. In certain embodiments, the Fab is fused to the N-terminus of the IgG, e.g., to the heavy chain or light chain N-terminus, optionally via a linker.
  • the Fab is fused to the N-terminus of the heavy chain of the IgG and not fused to the light chain.
  • the two heavy chains can be fused together directly or via a linker.
  • two or more VHH or sdAb may be fused together, optionally via a linker moiety, and fused to the Fab or IgG at one or more of these locations.
  • the Engineered WNT agonist has a Hetero- IgG format, whereas one of the Fab is present as a half antibody, and the other Fab is fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, or the C-terminus of the Fc.
  • the Fab or antigen-binding fragment thereof is fused directly to the other Fab or IgG or antigen-binding fragment thereof, whereas in other embodiments, the binding regions are fused via a linker moiety.
  • the WNT agonists of the present invention can have, comprise, or consist of any of the sequences provided in in any of the Tables, Figures, or Examples herein, or functional fragments or variants thereof.
  • the FZD binding domain, LRP5/6 binding domain, and/or engineered WNT agonist binds with a dissociation constant (KD) of about 1 mM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, or about 10 nM or less.
  • KD dissociation constant
  • a FZD binding domain or antibody described herein that binds to more than one FZD binds to those FZDs with a KD of about lOOnM or less, about 20 nM or less, or about 10 nM or less.
  • the binding domain binds to one or more its target antigen with an EC50 of about 1 mM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM 20 or less
  • the engineered WNT agonists, binding domains thereof, antibodies or other agents of the present invention can be assayed for specific binding by any method known in the art.
  • the immunoassays which can be used include, but are not limited to, competitive and noncompetitive assay systems using techniques such as BIAcore analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blots, radioimmunoassays, ELISA, "sandwich” immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays.
  • an ELISA assay comprises preparing antigen, coating wells of a 96 well microtiter plate with antigen, adding the antibody or other binding agent conjugated to a detectable compound such as an enzymatic substrate (e.g., horse-radish peroxidase or alkaline phosphatase) to the well, incubating for a period of time and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horse-radish peroxidase or alkaline phosphatase)
  • the antibody or agent is not conjugated to a detectable compound, but instead a second conjugated antibody that recognizes the first antibody or agent is added to the well.
  • the antibody or agent can be coated to the well and a second antibody conjugated to a detectable compound can be added following the addition of the antigen to the coated well.
  • a detectable compound can be added following the addition of the antigen to the coated well.
  • the binding affinity of an antibody or other agent to a target antigen and the off- rate of the antibody-antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., FZD, LRP), or fragment or variant thereof, with the antibody of interest in the presence of increasing amounts of unlabeled antigen followed by the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody and the binding off-rates can be determined from the data by scatchard plot analysis.
  • BIAcore kinetic analysis is used to determine the binding on and off rates of antibodies or agents.
  • BIAcore kinetic analysis comprises analyzing the binding and dissociation of antibodies from chips with immobilized antigens on their surface.
  • Engineered WNT agonists of the present invention are biologically active in binding to one or more FZD receptor and to one or more of LRP5 and LRP6, and in activation of WNT signaling.
  • WNT agonist activity refers to the ability of an agonist to mimic the effect or activity of a WNT protein binding to a frizzled protein and/or LRP5 or LRP6.
  • the ability of the engineered WNT agonists disclosed herein to mimic the activity of WNT can be confirmed by a number of assays.
  • WNT agonists typically initiate a reaction or activity that is similar to or the same as that initiated by the receptor's natural ligand.
  • the WNT agonists disclosed herein activate, enhance or increase the canonical WNT/ b-catenin signaling pathway.
  • the term “enhances” refers to a measurable increase in the level of WNT/ b-catenin signaling compared with the level in the absence of a WNT agonist, e.g., an engineered WNT agonist disclosed herein.
  • the increase in the level of WNT/ b-catenin signaling is at least 10%, at least 20%, at least 50%, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, atleast 1.5-fold, at least two-fold, atleast fivefold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to the level of WNT ⁇ -catenin signaling in the absence of the engineered WNT agonist, e.g., in the same cell type.
  • Methods of measuring WNTY b-catenin signaling are known in the art and include those described herein.
  • engineered WNT agonists disclosed herein are bispecific, i.e., they specifically bind to two or more different epitopes, e.g. , one or more FZD receptor, and LRP5 and/or LRP6. In certain embodiments, the engineered WNT agonists bind to FZD5 and/or FZD8, and LRP5 and/or LRP6.
  • engineered WNT agonists disclosed herein are multivalent, e.g., they comprise two or more regions that each specifically bind to the same epitope, e.g., two or more regions that bind to an epitope within one or more FZD receptor and/or two or more regions that bind to an epitope within LRP5 and/or LRP6. In particular embodiments, they comprise two or more regions that bind to an epitope within one or more FZD receptor and two or more regions that bind to an epitope within LRP5 and/or LRP6.
  • engineered WNT agonists comprise a ratio of the number of regions that bind one or more FZD receptor to the number of regions that bind LRP5 and/or LRP6 of or about: 1:1, 2:1, 3:1, 4:1, 5: 1, 6:1, 2:3, 2:5, 2:7, 7:2, 5:2, 3:2, 3:4, 3:5, 3:7, 3:8, 8:3, 7:3, 5:3, 4:3,
  • Engineered WNT agonists are bispecific and multivalent.
  • the present disclosure provides novel tissue-specific WNT signal enhancing molecules capable of enhancing WNT activity in a tissue- or cell-specific manner. These may be used alone or in combination with one or more engineered WNT agonist disclosed herein.
  • the tissue-specific WNT signal enhancing molecules are bi-functional molecules comprising a first domain that binds to one or more ZNRF3 and/or RNF43 ligases, and a second domain that binds to one or more targeted tissue or cell type in a tissue- or cell-specific manner.
  • Each of the first domain and the second domain may be any moiety capable of binding to the ligase complex or targeted tissue or cell, respectively.
  • each of the first domain and the second domain may be, but are not limited to, a moiety selected from: a polypeptide (e.g, an antibody or antigen-binding fragment thereof or a peptide or polypeptide different from an antibody), a small molecule, and a natural ligand or a variant, fragment or derivative thereof.
  • the natural ligand is a polypeptide, a small molecule, an ion, an amino acid, a lipid, or a sugar molecule.
  • the first domain and the second domain may be the same type of moiety as each other, or they may be different types of moieties.
  • the tissue-specific WNT signal enhancing molecules bind to a tissue- or cell-specific cell surface receptor.
  • the tissue-specific WNT signal enhancing molecules increase or enhance WNT signaling by at least 50%, at least two-fold, at least three-fold, at least five-fold, at least ten-fold, at least twenty-fold, at least thirty -fold, at least forty-fold, or at least fifty-fold, e.g., as compared to a negative control
  • Tissue-specific WNT signal enhancing molecules may have different formats.
  • the tissue-specific WNT signal enhancing molecules are fusion proteins comprising a first polypeptide sequence that binds to ZNRF3/RNF43 and a second polypeptide sequence that binds to one or more targeted tissue or cell type in a tissue- or cell- specific manner.
  • the two polypeptide sequences may be fused directly or via a linker.
  • the tissue-specific WNT signal enhancing molecules comprise two or more polypeptides, such as dimers or multimers comprising two or more fusion proteins, each comprising the first domain and the second domain, wherein the two or more polypeptides are linked, e.g., through a linker moiety or via a bond between amino acid residues in each of the two or more polypeptides, e.g., an intermolecular disulfide bond between cysteine residues.
  • a tissue-specific WNT signal enhancing molecule is an antibody comprising antibody heavy and light chains (or antigen-binding fragments thereof) that constitute either the first domain or the second domain, wherein the other domain (i.e., the second domain or first domain) is linked to the antibody heavy chain or light chain, either as a fusion protein or via a linker moiety.
  • the other domain is linked to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, or the C-terminus of the light chain.
  • Such structures may be referred to herein as appended IgG scaffolds or formats.
  • a tissue-specific WNT signal enhancing molecule can be an antibody that binds ZNRF3/RNF43, wherein a binding domain that binds a tissue- or cell-specific receptor is fused or appended to either the heavy chain or light chain of the antibody that binds ZNRF3/RNF43.
  • a tissue-specific WNT signal enhancing molecule can be an antibody that binds a tissue- or cell-specific receptor, wherein a binding domain that binds ZNRF3/RNF43 is fused or appended to either the heavy chain or light chain of the antibody that binds the tissue- or cell-specific receptor.
  • an intestine-specific WNT signal enhancing molecule is an antibody or antigen-binding fragment thereof that binds GPA33, CDH17, MUC-13, wherein a binding domain that binds ZNRF3/RNF43 is fused or appended to either the heavy chain or light chain of the antibody or antigen-binding fragment thereof.
  • the binding domain that bind ZNRF3/RNF43 comprises Ful and Fu2 domains, wherein the Ful and Fu2 domains optionally comprise one or more amino acid modifications, including any of those disclosed herein, e.g., F105R and/or F109A.
  • the tissue-specific WNT signal enhancing molecules comprise a first domain (“action module”) that binds ZNRF3/RNF43 and a second domain (“targeting module”) that binds a tissue- or cell-specific receptor, e.g., with high affinity.
  • each of these two domains has substantially reduced activity or is inactive in enhancing WNT signals by itself.
  • E3 ligases ZNRF3/RNF43 are recruited to a ternary complex with the tissue-specific receptors, leading them to be sequestered, and/or cleared from the cell surface via receptor-mediated endocytosis. The net result is to enhance WNT signals in a tissue-specific manner.
  • the action module is a binder to ZNRF3/RNF43 E3 ligases, and it can be designed based on R-spondins, e.g., R-spondins-1-4, including but not limited to human R-spondins-1-4.
  • the action module is an R-spondin, e.g., a wild-type R-spondin- 1-4, optionally a human R-spondin- 1-4, or a variant or fragment thereof.
  • the action module comprises or consists of a Furin domain 1 of an R-spondin, e.g., any of R-spondins 1-4, which bind ZNRF3/RNF43.
  • Extended versions of Furin domain 1 (including, but not limited to, those with a mutated Furin domain 2 that no longer binds to LGR4-6 or has reduced binding to LGR4-6) or engineered antibodies or any other derivatives or any engineered polypeptides different from antibodies that are able to bind specifically to ZNRF3/RNF43 can also be used.
  • the action module comprises one or more Furin domain 1 of an R- spondin.
  • the action module does not comprise a Furin domain 2 of an R-spondin, or it comprises a modified or variant Furin domain 2 of an R-spondin, e.g., a Furin domain 2 with reduced activity as compared to the wild-type Furin domain 2.
  • an action module comprises a Furin domain 1 but not a Furin domain 2 of R- spondin.
  • an action module comprises two or more Furin domain 1 or multimers of a Furin domain 1.
  • the action domain may comprise one or more wild-type Furin domain 1 of an R-spondin.
  • the action module comprises a modified or variant Furin domain 1 of an R-spondin that has increased activity, e.g., binding to ZNRF3/RNF43, as compared to the wild-type Furin domain 1.
  • Variants having increased binding to ZNRF3/RNF43 may be identified, e.g., by screening a phage or yeast display library comprising variants of an R-spondin Furin domain 1.
  • Peptides or polypeptides unrelated to R- spondin Furin domain 1 but with increased binding to ZNRF3/RNF43 may also be identified through screening.
  • Action modules may further comprise additional moieties or polypeptide sequences, e.g., additional amino acid residues to stabilize the structure of the action module or tissue-specific WNT signal enhancing molecule in which it is present.
  • the action module comprises another inhibitory moiety, such as a nucleic acid molecule, which reduces or prevents ZNRF3/RNF43 activity or expression, such as, e.g., an anti-sense oligonucleotide; a small interfering RNA (siRNA); a short hairpin RNA (shRNA); a microRNA (miRNA); or a ribozyme.
  • an anti-sense oligonucleotide such as, e.g., an anti-sense oligonucleotide; a small interfering RNA (siRNA); a short hairpin RNA (shRNA); a microRNA (miRNA); or a ribozyme.
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • ribozyme ribozyme
  • antisense RNA refers to single-stranded RNA molecules that can be introduced to an individual cell, tissue, or subject and results in decreased expression of a target gene through mechanisms that do not necessarily rely on endogenous gene silencing pathways.
  • An antisense nucleic acid can contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or others known in the art, or may contain non-natural intemucleoside linkages.
  • Antisense nucleic acid can comprise, e.g., locked nucleic acids (LNA).
  • the other inhibitor moiety inhibits an activity of one or both of ZNRF3/RNF43, or it inhibits the gene, mRNA or protein expression of one or both of ZNRF3/RNF43.
  • the inhibitory moiety is a nucleic acid molecule that binds to a ZNRF3/RNF43 gene or mRNA, or a complement thereof.
  • the targeting module specifically binds to a cell-specific surface molecule, e.g., a cell-specific surface receptor, and can be, e.g., natural ligands, antibodies, or synthetic chemicals.
  • the cell-specific surface molecule is preferentially expressed on a target organ, tissue or cell type, e.g., an organ, tissue or cell type in which it is desirous to enhance WNT signaling, e.g. , to treat or prevent a disease or disorder.
  • the cell-specific surface molecule has increased or enhanced expression on a target organ, tissue or cell type, e.g., an organ, tissue or cell type in which it is desirous to enhance WNT signaling, e.g., to treat or prevent a disease or disorder, e.g., as compared to one or more other non-targeted organs, tissues or cell types.
  • the cell-specific surface molecule is preferentially expressed on the surface of the target organ, tissue or cell type as compared to one or more other organ, tissue or cell types, respectively.
  • a cell surface receptor is considered to be a tissue-specific or cell-specific cell surface molecule if it is expressed at levels at least two- fold, at least five-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold higher in the target organ, tissue or cell than it is expressed in one or more, five or more, all other organs, tissues or cells, or an average of all other organs, tissue or cells, respectively.
  • the tissue-specific or cell-specific cell surface molecule is a cell surface receptor, e.g., a polypeptide receptor comprising a region located within the cell surface membrane and an extracellular region to which the targeting module can bind.
  • the methods described herein may be practiced by specifically targeting cell surface molecules that are only expressed on the target tissue or a subset of tissues including the target tissue, or by specifically targeting cell surface molecules that have higher levels of expression on the target tissue as compared to all, most, or a substantial number of other tissues, e.g., higher expression on the target tissue than on at least two, at least five, at least ten, or at least twenty other tissues.
  • Tissue-specific and cell-specific cell surface receptors are known in the art.
  • tissue- and cell-specific surface receptors include but are not limited to GPA33, CDH17, and MUC-13.
  • the targeting module comprises an antibody or antigen-binding fragment thereof that specifically binds these intestine specific receptors.
  • components of the engineered WNT agonist and WNT signal enhancing molecules may be combined to confer more tissue specificity.
  • the present invention is based, in part, upon the use of engineered WNT agonists to regulate gastrointestinal epithelium proliferation, in particular, in inflammatory bowel diseases.
  • the present invention provides a method of treating a subject suffering from a gastrointestinal disorder comprising administering to the subject, an engineered WNT signaling modulator.
  • the engineered WNT agonist comprises one or more binding domains that bind to one or more FZD receptors (FZDl-10) and one or more binding domains that bind to one or more LRP (LRP5-6) receptors.
  • the binding domains of the engineered WNT agonist comprise: one or more binding domains that bind to FZD5, FZD8, FZD1, FZD2, FZD7, FZD5,8, FZD 1,2, 7 or FZD 1,2, 7, 5, 8; FZD4; FZD9; or FZD 10; and one or more binding domains that bind to LRP5, LRP6, or LRP5 and 6.
  • the engineered WNT agonist comprises one or more binding domains that bind to FZD5 and/or FZD8; and one or more binding domains that bind to LRP5 and/or LRP6.
  • the engineered WNT agonist comprises a binding domain that binds to FZD5 and FZD8, and a binding domain that binds LRP6
  • the WNT agonist comprises a heavy chain sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17 or a variable heavy chain region derived therefrom; and a light chain sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18, or a variable light chain region derived therefrom.
  • the engineered WNT agonist comprises a tissue targeting molecule.
  • the tissue targeting molecule is an antibody or fragment thereof that binds to a tissue specific cell surface antigen.
  • the tissue targeting molecule is selected from the group consisting of Cell surface A33 antigen (GPA33; representative sequence is NCBI polypeptide reference sequence NP 005805.1), Cadherin-17 (CDH17; representative sequence is NCBI polypeptide reference sequence NP 004054.3), and Mucin 13 (cell surface associated (Muc-13; representative sequence is NCBI polypeptide reference sequence NP 149038.3), or a functional fragment or variant thereof.
  • the WNT agonist is administered with a binding domain that specifically binds an inflammatory molecule.
  • the binding domain specifically binding the inflammatory molecule is an antagonist of the inflammatory molecule.
  • the antagonist of the inflammatory molecule is an antagonist of TNFoc, IL-12, IL- 12 and IL-23, or IL-23.
  • the disclosure provides a combination molecule comprising: a) an engineered WNT agonist disclosed herein; and b) an engineered WNT signal enhancing molecule comprising a first domain that binds to one or more E3 ubiquitin ligases; and a second domain that binds to a tissue specific receptor.
  • the disclosure provides a polypeptide that specifically binds Frizzed 5 (FZD5) and Frizzled 8 (FZD8), wherein the polypeptide comprises one or more sequence having at least 80%, at least 90%, at least 95%, or at least 98% homology to a sequence set forth in any of SEQ ID NOs: 1-18.
  • the polypeptide comprises an antibody or antibody binding fragment, e.g., one or more variable heavy chain or variable light chain.
  • said antibody or antibody binding fragment comprises at least 5 or all six of the CDRs present in any of the following combinations of sequence: SEQ ID NOs: 1 and 2; SEQ IDNOs:3 and 4; SEQ ID NOs:5 and 6; or SEQ IDNOs:7 and 8, SEQ ID NOs:9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, or SEQ ID NOs: 17 and 18.
  • said polypeptide comprises six of the CDRs present in any of these combinations of sequences, wherein one or more of the CDRs comprises one, two, or three amino acid modifications, optionally a point mutation, an amino acid deletion, or an amino acid insertion.
  • the disclosure also provides a combination molecule comprising: an engineered WNT agonist disclosed herein; and an engineered WNT signal enhancing molecule comprising a first domain that binds to one or more E3 ubiquitin ligases; and a second domain that binds to a tissue specific receptor.
  • an engineered WNT agonist of the disclosure promotes cell differentiation (e g., gastrointestinal cells, stem cells, and/or epithelial cells) toward enterocytes.
  • cell differentiation is determined based on percentage of enterocyte precursors.
  • time-stamping-based methods are employed to determine cell differentiation toward enterocytes.
  • a lineage trajectory inference tool, slingshot is employed to complement timestamp-based observations of cell differentiation.
  • slingshot predicts the direction of cell differentiation from an initial starting group.
  • slingshot predicted cells would progress toward TA1, Goblet, Tufted, and enteroendocrine in one direction and toward enterocytes in the other directions.
  • predicted lineage trajectory pseudotime values show a higher percentage of engineered WNT agonist-treated samples that are further along in the enterocyte lineage trajectory relative to the control treated cells;
  • Figure 28E provides an illustrative example of predicted pseudotime values.
  • this prediction for the enterocyte lineage is congruent with the actual time-stamping data.
  • a reliable standard for validating improved differentiation is the expression of mature, differentiated cell type markers looked more like that of naive, uninjured colon in an engineered WNT agonist treatment group relative to the control groups on a given day after induced damage.
  • improved differentiation is observed 6-days after engineered WNT agonist treatment.
  • engineered WNT agonist treated samples include enterocytes, goblet cells, enteroendocrine, tuft cells, or a combination thereof.
  • Wnt mimetic molecules of the disclosure have desired properties, including the ability to restore diseased intestine tissue back to normal physiology.
  • short treatment using Wnt mimetics of the disclosure induces rapid restoration of epithelial tissue.
  • R2M13-26 or R2M13-h26 induces rapid restoration of epithelial tissue.
  • R2M13-26 in a severe DSS model, a single injection of R2M13-26 at various doses restored normal histology of the damaged colon tissue. Within 6 days of treatment, R2M13-h26 completely restored the epithelial barrier, which was severely damaged in the acute DSS model .
  • treatment withWnt mimetics of the disclosure reduces inflammatory cytokines and disease activity index, indicating elimination of the vicious cycle of barrier breach, microbial pathogen invasion, tissue inflammation and damage.
  • Wnt mimetics of the disclosure directly impact epithelial cells, expanding the progenitor pool and accelerating differentiation into all mature differentiated cell types.
  • Wnt mimetics of the disclosure restore Wnt signals and the stem cell niche in damaged colon tissue, without additional effects on the crypts after repair.
  • treatment with Wnt mimetics of the disclosure alone does not have effects on normal intestine epithelium.
  • RSPO may induce hyperplasia.
  • the disclosure provides a Wnt activator with optimal tissue repair and physiological activities.
  • R2M13-26 caused a robust increase in cell cycle gene expression in a broad spectrum of progenitor cells, whether normal stem/progenitors responding to injury or in altered cell states consistent with dedifferentiation.
  • These transcriptome changes manifested in the transient expansion of the progenitor pool and accelerated differentiation into the proper secretory and absorptive lineages of the colonic epithelium and re-establishment of the epithelial barrier. This direct impact on epithelial regeneration and barrier restoration secondarily led to a reduction in inflammatory signals and infiltrating immune cells.
  • the injury/damage context may set the stage for epithelial progenitor expansion.
  • injury caused an inflammatory response in all tissue layers.
  • interferon gamma and NF-KB pathways were active after injury, and recent work in other stem cell niches has shown that inflammatory signaling can facilitate the initial proliferative response to injury (M. Chen, Reed, & Lane, 2017; Kyritsis etah, 2012).
  • Activation of the NF-kB and Wnt pathways together may even promote the process of de-differentiation toward progenitors in the intestine (Schwitalla et al., 2013).
  • Wnt signaling was drastically reduced in the colonic epithelium possibly resulting from a reduction in expression of specific Wnts and an increase in several Wnt antagonists.
  • R2M13-h26 was able to overcome this Wnt signaling deficiency.
  • R2M13-h26-induced Wnt pathway activation may synergize with these inflammatory signals to enhance progenitor proliferation, albeit transiently.
  • Wnt pathway activation by R2M13-26 did not lead to crypt hyperproliferation or expansion. This stands in stark contrast to not only RSPO treatment but also the effects of heritable, genetic mutants.
  • the negative regulator Ape was genetically ablated or constitutively active mutants of Beta-catenin were expressed, crypts proliferated in an uncontrolled manner, failing to differentiate (Barker Nick et ah, 2009; Krausova & Korinek, 2014; Mah, Yan, & Kuo, 2016).
  • Wnt mimetics of the disclosure avoided these outcomes by mimicking endogenous Wnt signaling and initiating pathway activation at the receptor level, in contrast to the permanent genetic alterations that circumvent negative feedback.
  • R2M13- 26 By impacting the pathway at the level of the receptor, R2M13- 26 allowed negative feedback mechanisms to take effect.
  • Axin2 was induced, contributing to the destruction complex; expression of the E3 ubiquitin ligase Rnf43, also a Wnt target gene, was increased, which promotes the removal of FZD receptors from the cell surface.
  • R2M13-26 increased expression of some inhibitors of cyclin dependent kinases, potentially limiting proliferation.
  • compositions comprising an engineered WNT agonist molecule described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient are also disclosed.
  • the disclosure provides a pharmaceutical composition comprising a polypeptide, engineered WNT agonist, or combination molecule disclosed herein, and one or more pharmaceutically acceptable diluent, carrier, or excipient.
  • pharmaceutical compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a WNT agonist molecule (or a polypeptide chain thereof) described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient are also disclosed.
  • the polynucleotides are DNA or mRNA, e.g., a modified mRNA.
  • the polynucleotides are modified mRNAs further comprising a 5’ cap sequence and/or a 3’ tailing sequence, e.g ., a polyA tail.
  • the polynucleotides are expression cassettes comprising a promoter operatively linked to the coding sequences)).
  • the WNT agonist is an engineered recombinant polypeptide incorporating various epitope binding fragments that bind to various molecules in the WNT signaling pathway.
  • theFZD andLRP antibody fragments e.g., Fab, scFv, sdAbs, VHH, etc.
  • a polypeptide such as RSPO
  • RSPO may be engineered to contain an antibody or fragment thereof against a tissue specific cell surface antigen, e.g., MUC-13.
  • RSPO may also be administered concurrently or sequentially with an enhancer of the E3 ligases, ZNRF3/RNF43.
  • the E3 ligase enhancer may be an agonist antibody or fragment that binds ZNRF3/RNF43 and enhances the E3 ligase activity.
  • WNT agonists can also be recombinant polypeptides incorporating epitope binding fragments that bind to various molecules in the WNT signaling pathway and enhance WNT signaling.
  • a WNT agonist can be an antibody or fragment thereof that binds to FZD receptor and/or an LRP receptor and enhances WNT signaling.
  • the FZD and LRP antibody fragments e.g., Fab, scFv, sdAbs or VHHs, etc
  • compositions comprising an expression vector, e.g., a viral vector, comprising a polynucleotide comprising a nucleic acid sequence encoding a WNT agonist molecule described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient are also disclosed.
  • an expression vector e.g., a viral vector
  • a polynucleotide comprising a nucleic acid sequence encoding a WNT agonist molecule described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient
  • the present disclosure further contemplates a pharmaceutical composition
  • a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operatively linked to a nucleic acid encoding a WNT agonist molecule and one or more pharmaceutically acceptable diluent, carrier, or excipient.
  • the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide comprising a promoter operatively linked to a nucleic acid sequence encoding a WNT agonist.
  • the cell is a heterologous cell or an autologous cell obtained from the subject to be treated.
  • the subject molecules can be combined with pharmaceutically-acceptable carriers, diluents, excipients and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use.
  • excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations.
  • Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the pharmaceutical compositions are sterile.
  • compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition is sterile and may be fluid such that it can be drawn into a syringe or delivered to a subject from a syringe. In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi .
  • the carrier can be, e.g.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile solutions can be prepared by incorporating the engineered WNT agonist, e.g., an antibody or antigen-binding fragment thereof (or encoding polynucleotide or cell comprising the same) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by Filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the pharmaceutical compositions are prepared with carriers that will protect the antibody or antigen-binding fragment thereof against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active antibody or antigen-binding fragment thereof calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms are dictated by and directly dependent on the unique characteristics of the antibody or antigen-binding fragment thereof and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active antibody or antigen-binding fragment thereof for the treatment of individuals.
  • compositions can be included in a container, pack, or dispenser, e.g. syringe, e.g. a prefilled syringe, together with instructions for administration.
  • syringe e.g. a prefilled syringe
  • the pharmaceutical compositions of the present disclosure encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal comprising a human, is capable of providing (directly or indirectly) the biologically active antibody or antigen-binding fragment thereof.
  • the present disclosure includes pharmaceutically acceptable salts of a WNT agonist molecule described herein.
  • pharmaceutically acceptable salt refers to physiologically and pharmaceutically acceptable salts of the compounds of the present disclosure: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • a variety of pharmaceutically acceptable salts are known in the art and described, e.g., in “Remington’s Pharmaceutical Sciences”, 17th edition, Alfonso R.
  • Metals used as cations comprise sodium, potassium, magnesium, calcium, and the like.
  • Amines comprise N-N’-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N- methylglucamine, and procaine (see, for example, Berge et ab, “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119).
  • the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
  • the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present disclosure.
  • the pharmaceutical composition provided herein comprise a therapeutically effective amount of a WNT agonist molecule or pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable carrier, diluent and/or excipient, for example saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives and other proteins.
  • a pharmaceutically acceptable carrier for example saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives and other proteins.
  • Exemplary amino acids, polymers and sugars and the like are octylphenoxy polyethoxy ethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycol.
  • the pharmaceutical composition provided herein comprises a buffer, such as phosphate buffered saline (PBS) or sodium phosphate/sodium sulfate, tris buffer, glycine buffer, sterile water and other buffers known to the ordinarily skilled artisan such as those described by Good et al. (1966) Biochemistry 5:467
  • PBS phosphate buffered saline
  • tris buffer such as sodium phosphate/sodium sulfate
  • glycine buffer sodium phosphate/sodium sulfate
  • glycine buffer such as those described by Good et al. (1966) Biochemistry 5:467
  • the pH of the buffer may be in the range of 6.5 to 775, preferably 7 to 7 5, and most preferably 7.2 to 7.4.
  • the present disclosure also provides methods for using the engineered WNT agonists and/or tissue-specific WNT signal enhancing molecules, e.g., to modulate a WNT signaling pathway, e.g., to increase WNT signaling, and the administration of an engineered WNT agonist and/or tissue-specific WNT signal enhancing molecule in a variety of therapeutic settings.
  • an engineered WNT agonist may be used to increase Wnt signaling in a tissue or cell.
  • the present invention provides a method for increasing Wnt signaling or enhancing Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of an engineered WNT agonist or pharmaceutically acceptable salt thereof disclosed herein, wherein the an engineered WNT agonist is a Wnt signaling pathway agonist.
  • contacting occurs in vitro, ex vivo, or in vivo.
  • the cell is a cultured cell, and the contacting occurs in vitro.
  • the method comprises further contacting the tissue or cell with one or more Wnt polypeptides or Norrin polypeptides.
  • Engineered WNT agonists disclosed herein may be used in to treat a disease, disorder or condition, for example, by increasing Wnt signaling in a targeted cell, tissue or organ
  • the present invention provides a method for treating a disease or condition in a subject in need thereof, e.g., a disease or disorder associated with reduced Wnt signaling, or for which increased Wnt signaling would provide a therapeutic benefit, comprising contacting the subject with an effective amount of a composition of the present disclosure.
  • the composition is a pharmaceutical composition comprising any of: an engineered WNT agonist; a polynucleotide comprising a nucleic acid sequence encoding an engineered WNT agonist, e.g., a DNA or mRNA, optionally a modified mRNA; a vector comprising a nucleic acid sequence encoding an engineered WNT agonist, e.g., an expression vector or viral vector; or a cell comprising a nucleic acid sequence encoding a an engineered WNT agonist.
  • the disease or condition is a pathological disease or disorder, or an injury, e.g., an injury resulting from a wound.
  • the wound may be the result of another therapeutic treatment.
  • the disease or condition comprises impaired tissue repair, healing or regeneration, or would benefit from increased tissue repair, healing or regeneration.
  • contacting occurs in vivo, i.e , the subject composition is administered to a subject.
  • Wnt signaling plays key roles in the developmental process and maintenance of stem cells. Reactivation of Wnt signals is associated with regeneration and repair of most tissues after injuries and diseases. Engineered WNT agonist molecules are expected to provide benefit of healing and tissue repair in response to injuries and diseases. Causes of tissue damage and loss include but are not limited to aging, degeneration, hereditary conditions, infection and inflammation, traumatic injuries, toxins/metabolic-induced toxicities, or other pathological conditions. Wnt signals and enhancers of Wnt signals have been shown to activate adult, tissue- resident stem cells. In some embodiments, the compounds of the invention are administered for use in treating diseased or damaged tissue, for use in tissue regeneration and for use in cell growth and proliferation, and/or for use in tissue engineering.
  • compositions of the present invention may be used to promote or increase bone growth or regeneration, bone grafting, healing of bone fractures, treatment of osteoporosis and osteoporotic fractures, spinal fusion, spinal cord injuries, including vertebral compression fractures, pre-operative spinal surgery optimization, osseointegration of orthopedic devices, tendon-bone integration, tooth growth and regeneration, dental implantation, periodontal diseases, maxillofacial reconstruction, and osteonecrosis of the jaw. They may also be used in the treatment of alopecia; enhancing regeneration of sensory organs, e.g.
  • treatment of hearing loss including regeneration of inner and outer auditory hair cells treatment of vestibular hypofunction, treatment of macular degeneration, treatment of retinopathies, including vitreoretinopathy, diabetic retinopathy, other diseases of retinal degeneration, Fuchs’ dystrophy, other cornea disease, etc.; treatment of stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, multiple dystrophy, muscle atrophy as a result of sarcopenia or cachexia, and other conditions affecting the degeneration or integrity of the blood brain barrier.
  • the present invention provides a method for treating a subject having a disease or disorder associated with reduced WNT signaling or for which increased Wnt signaling may be beneficial, comprising administering to the subject an effective amount of an engineered WNT agonist, or a pharmaceutical composition comprising an engineered WNT agonist.
  • the disease or disorder is selected from the group consisting of: oral mucositis, short bowel syndrome, inflammatory bowel diseases (IBD), other gastrointestinal disorders, including, but not limited to: graft versus host disease (GVHD), alcoholic hepatitis, short bowel syndrome, celiac disease, radiation-induced gastrointestinal mucositis and chemotherapy-induced gastro-intestinal mucositis; treatment of metabolic syndrome, dyslipidemia, treatment of diabetes, treatment of pancreatitis, conditions where exocrine or endocrine pancreas tissues are damaged; conditions where enhanced epidermal regeneration is desired, e.g., epidermal wound healing, treatment of diabetic foot ulcers, syndromes involving tooth, nail, or dermal hypoplasia, etc., conditions where angiogenesis is beneficial; myocardial infarction, coronary artery disease, heart failure; immunodeficiencies, graft versus host diseases, acute kidney injuries, chronic kidney diseases, chronic obstructive pulmonary diseases (COPD),
  • COPD chronic
  • the engineered WNT agonists and compositions of this invention may also be used in treatment of oral mucositis, treatment of short bowel syndrome, inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), in particular CD with fistula formation, other gastrointestinal disorders; treatment of metabolic syndrome, dyslipidemia, treatment of diabetes, treatment of pancreatitis, conditions where exocrine or endocrine pancreas tissues are damaged; conditions where enhanced epidermal regeneration is desired, e.g., epidermal wound healing, treatment of diabetic foot ulcers, syndromes involving tooth, nail, or dermal hypoplasia, etc., conditions where angiogenesis is beneficial; treatment of myocardial infarction, coronary artery disease, heart failure; enhanced growth of hematopoietic cells, e.g.
  • compositions of the present invention may also be used in enhanced regeneration of liver cells, e.g.
  • compositions of this invention may treat diseases and disorders including, without limitation, conditions in which regenerative cell growth is desired.
  • bone regeneration is enhanced by contacting a responsive cell population, e.g. bone marrow, bone progenitor cells, bone stem cells, etc. with an effective dose of an engineered WNT agonist disclosed herein.
  • Methods for regeneration of bone tissues benefit from administration of the engineered WNT agonist which can be systemic or localized.
  • the contacting is performed in vivo
  • the contacting is performed ex vivo.
  • the molecule may be localized to the site of action, e.g. by loading onto a matrix, which is optionally biodegradable, and optionally provides for a sustained release of the active agent.
  • Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, polymeric microspheres, nanoparticles, bone cements, and the like
  • compositions comprising one or more engineered WNT agonist disclosed herein (or a polynucleotide encoding an engineered WNT agonist, or a vector or cell comprising a polynucleotide encoding a Engineered WNT agonist) are used to treat or prevent a bone disease or disorder, including but not limited to any of the following, or to treat or prevent an injury associated with, but not limited to, any of the following: osteoporosis, osteoporotic fractures, bone fractures including vertebral compression fractures, non-union fractures, delayed union fractures, spinal fusion, osteonecrosis, osteonecrosis of the jaw, hip, femoral head, etc., osseointegration of implants (e.g., to accelerate recovery following partial or total knee or hip replacement), osteogenesis imperfecta, bone grafts, tendon repair, maxillofacial surgery, dental implant, all other bone disorders or defects resulting from genetic diseases, degeneration, aging,
  • a bone disease or disorder
  • engineered WNT agonists that bind Fzdl, Fzd 2, and Fzd 7, and also LRP5 and/or LRP6, are used to treat or prevent any bone disease or disorder.
  • Engineered WNT agonists that bind Fzdl, Fzd 2, Fzd 5, Fzd 7 and Fzd 8, and also LRP5 and/or LRP6, are used to treat or prevent any bone disease or disorder.
  • Other Fzd molecules that bind to additional Fzd receptors can also be used with LRP5 and/or LRP6 binders.
  • compositions and methods disclosed herein may be used to: increase bone mineral density, increase bone volume (e.g. , tibia and/or femur bone volume), increase cortical thickness (e.g., in trabecular region or in femur mid-diaphysis), increase mineral apposition rate, increase the number of osteblasts and/or decrease the number of osteoclasts (e.g., in bone), increase bone stiffness, increase the ultimate load to fracture point, improve bone resistance to fracture, decrease bone resorption, decrease bone loss associated with osteoporosis, or increase biochemical strength of bone, in a subject.
  • bone volume e.g. , tibia and/or femur bone volume
  • cortical thickness e.g., in trabecular region or in femur mid-diaphysis
  • increase mineral apposition rate e.g., in trabecular region or in femur mid-diaphysis
  • engineered WNT agonists that bind Fzdl, Fzd 2, and Fzd 7 are used for any of these indicated uses. In one embodiment, engineered WNT agonists that bind Fzdl, Fzd 2, Fzd 5, Fzd 7 and Fzd 8 are used for any of these indicated uses.
  • Methods disclosed herein, including methods for treating or preventing a bone disease or disorder include methods that comprise providing to a subject in need thereof both an engineered WNT agonist and an antiresorptive agent. In certain embodiments, the methods are used for the treatment of osteoporosis, optionally post-menopausal osteoporosis.
  • the disclosure also provides a method for inhibiting or reducing bone resorption in a subject in need thereof, comprising providing to the subject an effective amount of an engineered WNT agonist, wherein the engineered WNT agonist is an agonist of a Wnt signaling pathway. In certain embodiments, the method further comprises providing to the subject an antiresorptive agent.
  • the subject has been diagnosed with or is at risk for osteoporosis, optionally postmenopausal osteoporosis
  • osteoporosis optionally postmenopausal osteoporosis
  • antiresorptive agents include, but are not limited to, those disclosed herein.
  • the two agent may be provided in the same or different pharmaceutical compositions. They may be provided to the subject at the same time, at different times, e.g. , simultaneously, consecutively, or during overlapping or nonoverlapping time periods. In certain embodiments, the two agents are therapeutically active in the subject during an overlapping time period
  • compositions comprising one or more engineered WNT agonist disclosed herein (or a polynucleotide encoding an engineered WNT agonist, or a vector or cell comprising a polynucleotide encoding an engineered WNT agonist) can be used for the in vivo treatment of skeletal tissue deficiencies.
  • skeletal tissue deficiency it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e.g., whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions.
  • compositions of the present invention can be used as part of a regimen for restoring cartilage function to a connective tissue, for the repair of defects or lesions in cartilage tissue such as degenerative wear and arthritis, trauma to the tissue, displacement of tom meniscus, meniscectomy, a luxation of ajoint by a torn ligament, malalignment of joints, bone fracture, or by hereditary disease.
  • An engineered WNT agonist may also be used for treatment of periodontal diseases.
  • Periodontal diseases are a leading cause of tooth loss and are linked to multiple systemic conditions.
  • tooth or underlying bone regeneration is enhanced by contacting a responsive cell population.
  • the contacting is performed in vivo.
  • the contacting is performed ex vivo, with subsequent implantation of the activated stem or progenitor cells.
  • the molecule may be localized to the site of action, e.g., by loading onto a matrix, which is optionally biodegradable, and optionally provides for a sustained release of the active agent.
  • Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymeric microspheres, nanoparticles, and the like.
  • compositions of the invention are capable of promoting sensory hair cell regeneration in the inner ear following injuries, aging, or degeneration. Loss of sensory hair cells in the inner ear involved in hearing loss or vestibular hypofunction may also benefit from the compositions of the invention.
  • the auditory organ houses mechanosensitive hair cells required for translating sound vibration to electric impulses.
  • the vestibular organs comprised of the semicircular canals (SSCs), the utricle, and the saccule, also contain sensory hair cells in order to detect head position and motion.
  • Compositions of the present invention can be used, for example, in an infusion, in a matrix or other depot system; or other topical application to the ear for enhancement of auditory regeneration.
  • An engineered WNT agonist may also be used in regeneration of retinal tissue.
  • Muller glia cells are capable of regenerating retinal cells, including photoreceptors, for example after neurotoxic injury in vivo.
  • Wnt signaling and enhancers of Wnt signals can promote proliferation of Muller glia-derived retinal progenitors after damage or during degeneration.
  • the compositions of the invention may also be used in the regeneration of tissues and other cell types in the eye. For examples age-related macular degeneration (AMD), other retina degenerative diseases, cornea diseases, Fuchs’ dystrophy, vitreoretinopathy, hereditary diseases, etc. can benefit from the compositions of the present inventions.
  • AMD age-related macular degeneration
  • AMD is characterized by progressively decreased central vision and visual acuity.
  • Fuchs’ dystrophy is characterized by progressive loss of cornea endothelial cells.
  • Wnt signal and enhancing of Wnt signal can promote regeneration of cornea endothelium, retina epithelium, etc. in the eye tissue.
  • compositions of the present invention can be used, for example, in an infusion; in a matrix or other depot system; or other topical application to the eye for retinal regeneration and treatment of macular degeneration.
  • compositions comprising an engineered WNT agonist disclosed herein (or a polynucleotide encoding an engineered WNT agonist, or a vector or cell comprising a polynucleotide encoding an engineered WNT agonist) are used to promote liver regeneration, reduce fibrosis, and/or improve liver function.
  • compositions and methods disclosed herein are used to: increase liver weight, increase the liver to body weight ratio, increase the number of PCNA and pH3 positive nuclei in liver, increase expression of Ki67 and/or Cyclin D1 in liver, increase liver cell proliferation and/or mitosis, decrease fibrosis following chronic liver injury, or increase hepatocyte function.
  • compositions of this invention may be used in treatment of acute liver failure, acute alcoholic liver injuries, treatment of chronic liver diseases with hepatitis C or B virus infection or post-antiviral drug therapies, chronic alcoholic liver diseases, alcoholic hepatitis, non-alcoholic fatty liver diseases and non-alcoholic steatohepatitis (NASH), treatment of cirrhosis and severe chronic liver diseases of all causes, and enhanced regeneration of liver cells.
  • Methods for regeneration of liver tissue benefit from administration of the compounds of the invention, which can be systemic or localized These include, but are not limited to, methods of systemic administration and methods of localized administration e.g. by injection into the liver tissue, by injection into veins or blood vessels leading into the liver, by implantation of a sustained release formulation, and the like.
  • compositions comprising an engineered WNT agonist disclosed herein (or a polynucleotide encoding an engineered WNT agonist, or a vector or cell comprising a polynucleotide encoding an engineered WNT agonist) are used to treat or prevent a liver disease or disorder, including but not limited to, or to treat or prevent a liver injury or disorder resulting from any of the following: acute liver failure (all causes), chronic liver failure (all causes), cirrhosis, liver fibrosis (all causes), portal hypertension, alcoholic liver diseases including alcoholic hepatitis, nonalcoholic steatohepatisis (NASH), nonalcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, hepatitis C virus-induced liver diseases (HCV), hepatitis B virus-induced liver diseases (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver diseases (HAV) and
  • Various epidermal conditions benefit from treatment with the compounds of the present invention. Mucositis occurs when there is a breakdown of the rapidly divided epithelial cells lining the gastro-intestinal tract, leaving the mucosal tissue open to ulceration and infection.
  • the part of the epithelial lining that covers the mouth, called the oral mucosa is one of the most sensitive parts of the body and is particularly vulnerable to chemotherapy and radiation.
  • Oral mucositis is probably the most common, debilitating complication of cancer treatments, particularly chemotherapy and radiation
  • the compositions of the invention may also benefit treatment of short bowel syndrome, inflammatory bowel diseases (IBD), or other gastrointestinal disorders.
  • IBD inflammatory bowel diseases
  • epidermal conditions include epidermal wound healing, diabetic foot ulcers, syndromes involving tooth, nail, or dermal hypoplasia, and the like. Molecules of the present invention may be used in all these conditions, where regenerative cells are contacted with compounds of the invention. Methods for regeneration of epithelial tissues benefit from administration of the compounds of the invention, which can be systemic or localized. Contacting can be, for example, topical, including intradermal, subdermal, in a gel, lotion, cream etc. applied at targeted site, etc.
  • Wnt signals and enhancement and promotion of Wnt signals also play an important role in repair and regeneration of tissues including pancreas, kidney, and lung in preclinical models.
  • An engineered WNT agonist may benefit various disease conditions involving exocrine and endocrine pancreas, kidney, or lung.
  • the engineered WNT agonists may be used in treatment of metabolic syndrome; treatment of diabetes, treatment of acute or chronic pancreatitis, exocrine pancreatic insufficiency, treatment of acute kidney injuries, chronic kidney diseases, treatment of lung diseases, including but not limited to chronic obstructive pulmonary diseases (COPD), pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), and other conditions that cause loss of lung epithelial tissues
  • COPD chronic obstructive pulmonary diseases
  • IPF idiopathic pulmonary fibrosis
  • Methods for regeneration of these tissues benefit from administration of the compounds of the invention, which can be systemic or localized.
  • hair follicle regeneration is enhanced by contacting a responsive cell population with a molecule of the present invention.
  • the contacting is performed in vivo.
  • the contacting is performed ex vivo.
  • the molecule may be localized to the site of action, e.g.
  • BBB blood brain barrier
  • Angiogenesis is critical to ensure the supply of oxygen and nutrients to many tissues throughout the body, and is especially important for the CNS as the neural tissue is extremely sensitive to hypoxia and ischemia.
  • CNS endothelial cells which form the BBB differ from endothelial cells in non-neural tissue, in that they are highly polarized cells held together by tight junctions and express specific transporters. Wnt signaling regulates CNS vessel formation and/or function.
  • Conditions in which the BBB is compromised can benefit from administration of the compounds of the invention, which can be systemic or localized e.g. by direct injection, intrathecal administration, implantation of sustained release formulations, and the like.
  • Wnt signal is actively involved in neurogenesis and plays a role of neuroprotection following injury.
  • the compositions of the present invention may also be used in treatment of spinal cord injuries, other spinal cord diseases, stroke, traumatic brain injuries, etc.
  • Wnt signals also play a role in angiogenesis.
  • An engineered WNT agonist may benefit conditions where angiogenesis is beneficial, treatment of myocardial infarction, coronary artery disease, heart failure, diabetic retinopathy, etc., and conditions from hereditary diseases. Methods for regeneration of these tissues benefit from administration of the compounds of the invention, which can be systemic or localized.
  • methods of the present invention promote tissue regeneration, e.g., in a tissue subjected to damage or tissue or cell reduction or loss.
  • the loss or damage can be anything which causes the cell number to diminish, including diseases or injuries.
  • an accident, an autoimmune disorder, a therapeutic side-effect or a disease state could constitute trauma.
  • Tissue regeneration increases the cell number within the tissue and preferably enables connections between cells of the tissue to be re-established, and more preferably the functionality of the tissue to be regained.
  • administering or “introducing” or “providing”, as used herein, refer to delivery of a composition to a cell, to cells, tissues and/or organs of a subject, or to a subject. Such administering or introducing may take place in vivo, in vitro or ex vivo.
  • a pharmaceutical composition is administered parenterally, e.g., intravenously, orally, rectally, or by injection. In some embodiments, it is administered locally, e.g., topically or intramuscularly.
  • a composition is administered to target tissues, e.g., to bone, joints, ear tissue, eye tissue, gastrointestinal tract, skin, a wound site or spinal cord. Methods of the invention may be practiced in vivo or ex vivo. In some embodiments, the contacting of a target cell or tissue with an engineered WNT agonist is performed ex vivo, with subsequent implantation of the cells or tissues, e.g., activated stem or progenitor cells, into the subject. The skilled artisan can determine an appropriate site of and route of administration based on the disease or disorder being treated.
  • the dose and dosage regimen may depend upon a variety of factors readily determined by a physician, such as the nature of the disease or disorder, the characteristics of the subject, and the subject's history.
  • the amount of an engineered WNT agonist administered or provided to the subject is in the range of about 0.01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 50 mg/kg of the subject’s body weight.
  • the WNT agonist is administered to a subject, e.g., a mammal, intravenously, e.g., as a bolus injection, or subcutaneously.
  • the WNT agonist is administered at least once per week.
  • the subject is administered about 0.5 to about 100 mg/kg body weight of the WNT agonist, or about 2 to about 50 mg/kg body weight of the WNT agonist, e.g., about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg.
  • the subject is administered about 25 mg, about 75 mg, about 250 mg, about 750 mg, about 1500 mg or about 2250 mg of the WNT agonist.
  • the subject is administered about 3 to about 30 mg/kg body weight intravenously or subcutaneously at least once per week of R2M13-h26, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO: 9 and two polypeptides of SEQ ID NO: 10 bound by disulfide bonds.
  • treatment used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, e.g. reducing the likelihood that the disease or symptom thereof occurs in the subject, and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent e.g., a Engineered WNT agonist
  • the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues.
  • the subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • the subject method results in a therapeutic benefit, e.g. , preventing the development of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc.
  • the subject method comprises the step of detecting that a therapeutic benefit has been achieved. The ordinarily skilled artisan will appreciate that such measures of therapeutic efficacy will be applicable to the particular disease being modified, and will recognize the appropriate detection methods to use to measure therapeutic efficacy.
  • the methods disclosed herein result in one or more of the following PK/PD parameters: Clearance (mL/day/kg) of 10-50 or about 25; terminal tl/2 of 2- 5 days or about 4 days; Cmax (ug/mL) of 50 to 300 or 100 to 200, or about 140, MRT of about 3 to 4 (days), or about 4, or AUC (day*pg/mL) of about 100 to 1000 or about 100 to about 500, or about 190.
  • Other embodiments relate, in part, to the use of the engineered WNT agonists disclosed herein to promote or enhance the growth or proliferation of cells, tissues and organoids, for example, by contacting cells or tissue with one or more engineered WNT agonist, optionally in combination with a Norrin or Rspondin polypeptide.
  • the cells or tissue are contacted ex vivo, in vitro, or in vivo.
  • Such methods may be used to generate cells, tissue or organoids for therapeutic use, e.g., to be transplanted or grafted into a subject. They may also be used to generate cells, tissue or organoids for research use.
  • the engineered WNT agonists have widespread applications in non-tlierapeutie methods, for example in vitro research methods.
  • the engineered WNT agonists may be used to preserve cells, tissues, organs or organoids, e.g., tissue or organs for transplantation.
  • a cell, tissue, organ, or organoid may be contacted with an engineered WNT agonist in vivo or ex vivo.
  • the cell, tissue, organ, or organoid may be contacted with an engineered WNT agonist while still in the donor ( i.e ., before removal from the donor) and/or after removal from the donor.
  • the methods may maintain or enhance viability of the cell, tissue, or organ, for example, during storage or prior to transplantation into a recipient.
  • the cells, tissue, or organ is perfused in a composition or solution comprising the engineered WNT agonist.
  • certain organ tissue is contacted with a WNT super agonist molecule to maintain viability of that tissue.
  • the organ tissue is donor organ tissue to be transplanted to a recipient in need thereof.
  • donor organ tissue is perfused in vivo with a solution comprising an engineered WNT agonist disclosed here, e.g.. before the organ tissue is removed from the donor
  • donor organ tissue is perfused ex vivo with a solution comprising an engineered WNT agonist disclosed here, e.g. , during storage or during transport from a donor to a recipient.
  • the organ tissue contacted with an engineered WNT agonist remains viable for transplantation for at least 10%, at least 20%, at least 50%, or at least 100% longer than if it was not contacted with the engineered WNT agonist.
  • the organ tissue is liver tissue.
  • the engineered WNT agonists may be used for the expansion and/or maintenance of ex vivo tissue, e.g., skin tissue.
  • tissue e.g., skin tissue.
  • the tissue is isolated from a donor or a patient.
  • the tissue may be contacted with (e.g., maintained or cultured in the presence of) an engineered WNT agonist in vivo or ex vivo.
  • the tissue is contacted ex vivo, e.g., by perfusion with a composition comprising an engineered WNT agonist.
  • the engineered WNT agonists may be used to generate or maintain an organoid or organoid culture.
  • an organoid culture may be contacted with an engineered WNT agonist, for example, by culturing the organoid in a medium comprising an engineered WNT agonist.
  • an organoid culture is generated, grown, or maintained by contacting it with one or more engineered WNT agonist disclosed herein.
  • the engineered WNT agonist is present in the culture media used to grow or maintain the organoid tissue.
  • the invention provides a method for tissue regeneration of damaged tissue, such as the tissues discussed above, comprising administering an engineered WNT agonist to cells.
  • the engineered WNT agonist may be administered directly to the cells in vivo, administered to a subject orally, intravenously, or by other methods known in the art, or administered to ex vivo cells.
  • these cells may be transplanted into a subject before, after or during administration of the engineered WNT agonist.
  • Wnt signaling is a key component of stem cell culture.
  • stem cell culture media as described in W 02010/090513, WO2012/014076, Sato et al., 2011 (GASTROENTEROLOGY 201 1, 141: 1762-1772) and Sato et ah, 2009 (Nature 459, 262-5).
  • the engineered WNT agonists disclosed herein are suitable alternatives to Rspondin for use in these stem cell culture media, or may be combined with Rspondin.
  • the disclosure provides a method for enhancing the proliferation of stem cells comprising contacting stem ceils with one or more Engineered WNT agonists disclosed herein, in one embodiment, the disclosure provides a cell culture medium comprising one or more engineered WNT agonists disclosed herein.
  • die ceil culture medium may be any cell culture medium already known in the art that normally comprises Writ or Rspondin, but wherein the Wnt or Rspondin is replaced (wholly or partially) or supplemented by engineered WNT agonist(s) disclosed herein.
  • the culture medium may be as described in as described in W02010/090513, WO2012/014076, Sato el a!., 2011 (GASTROENTEROLOGY 201 1, 141 : 1762-1772) and Sato et ah, 2009 (Nature 459, 262-5), which are hereby incorporated by reference in their entirety
  • Stem cell culture media often comprise additional growth factors This method may thus additionally comprise supplying the stem cells with a growth factor.
  • Growth factors commonly used in ceil culture medium include epidermal growth factor (EGF, (Peprotech), Transforming Growth Factor-alpha (TGF -alpha, Peprotech), basic Fibroblast Growth Factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), Hepatoeyte Growth Factor (HGF) and Keratinocyte Growth Factor (KGF, Peprotech, also known as FGF7)
  • EGF is a potent mitogenic factor for a variety of cultured ectodermal and mesodermal cells and has a profound effect, on the differentiation of specific cells in vivo and in vitro and of some fibroblasts in cell culture.
  • the EGF precursor exists as a membrane-bound molecule which is proteoiyticaliy cleaved to generate the 53-amino acid peptide hormone that stimulates cells EGF or other mitogenic growth factors may thus be supplied to the stem cells During culturing of stem cells, the mitogenic growth factor may be added to the culture medium every second day, while the culture medium is refreshed preferably every fourth day.
  • a mitogenic factor is selected from the groups consisting of: i) EGF, TGF-alpha, and KGF, ii) EGF, TGF-alpha, and FGF7; til) EGF, TGF-alpha, and FGF; iv) EGF and KGF; v) EGF and FGF7; vi) EGF and a FGF, vii) TGF-alpha and KGF; viii) TGF-alpha, and FGF7; ix) or from TGF-alpha and a FGF.
  • the disclosure includes a stem cell culture media comprising a Engineered WNT agonist disclosed herein, e.g., optionally in combination with one or more of the growth factors or combinations thereof described herein.
  • the engineered WNT agonists are used to enhance stem ceil regeneration.
  • Illustrative stem ceils of interest include but are not limited to: muscle satellite cells; hematopoietic stem cells and progenitor cells derived therefrom (U S Pat No. 5,061 ,620); neural stem cells (see Morrison et al. (1999) Cell 96: 737-749); embryonic stem cells; mesenchymal stem ceils; mesodermal stem ceils; liver stem cells, adipose-tissue derived stem ceils, etc
  • the present invention is based, in part, upon the use of engineered WNT agonists to regulate gastrointestinal epithelium proliferation, in particular, in inflammatory bowel diseases.
  • the present invention provides a method of treating a subject suffering from a gastrointestinal disorder comprising administering to the subject, an engineered WNT agonist disclosed herein.
  • the gastrointestinal disease is inflammatory bowel disease.
  • the inflammatory bowel disease is selected from the group consisting of: Crohn’s disease (CD), CD with fistula formation, and ulcerative colitis (UC).
  • the engineered WNT agonist reduces inflammatory cytokine expression in the intestine or colon and/or repairs intestinal epithelium.
  • the present invention also provides a method of treating a subject suffering from a gastrointestinal disorder comprising administering to the subject a tissue- specific WNT signal enhancing molecule.
  • the WNT signal enhancing molecule comprises: a) a first domain that binds to one or more E3 ubiquitin ligases; and b) a second domain that binds to a tissue specific receptor.
  • the E3 ubiquitin ligases are selected from the group consisting of Zinc and Ring Finger Protein 3 (ZNRF3) and Ring Finger Protein 43 (RNF43).
  • the first domain comprises an R-spondin (RSPO) polypeptide.
  • the RSPO polypeptide is selected from the group consisting of RSPO-1, RSPO-2, RSPO-3, and RSPO-4.
  • the RSPO polypeptide comprises a first furin domain and a second furin domain.
  • the second furin domain is wild-type or is mutated to have lower binding to Leucine-rich repeat-containing G protein coupled receptors 4-6 (LGR4-6).
  • the engineered agonist or Wnt signal enhancing molecule incorporates a tissue targeting molecule.
  • the tissue targeting molecule is an antibody or fragment thereof that binds to a tissue specific cell surface antigen.
  • the tissue targeting molecule is selected from the group consisting of GPA33, CDH17, and MUC- 13, or a functional fragment or variant thereof.
  • the WNT agonist is administered with a binding domain that specifically binds an inflammatory molecule.
  • the binding domain specific for the inflammatory molecule is an antagonist of the inflammatory molecule.
  • the antagonist of the inflammatory molecule is an antagonist of TNFa, EL-12, IL-12 and IL-23, or IL-23.
  • the gastrointestinal disease is inflammatory bowel disease.
  • the inflammatory bowel disease is selected from the group consisting of: Crohn’s disease (CD), CD with fistula formation, and ulcerative colitis (UC)
  • the present invention provides for a method of treating a subject suffering from a gastrointestinal disorder comprising administering to the subject an engineered WNT agonist and an engineered tissue specific WNT signal enhancing molecule.
  • the engineered WNT agonist and the engineered tissue specific WNT signal enhancing molecule may be administered at the same time or at different times.
  • the subject comprises an effective amount of both during an overlapping time period.
  • the engineered WNT agonist comprises one or more binding domains that bind toFZD5, FZD8, FZD1, FZD2, FZD7, FZD 5and 8, or FZD1, 2, and 7, and one or more binding domains that bind to LRP5, LRP6, or LRP5.
  • the engineered WNT agonist comprises a tissue targeting molecule
  • the tissue targeting molecule is an antibody or fragment thereof that binds to a tissue specific cell surface antigen.
  • the tissue targeting molecule is selected from the group consisting of GPA33, CDH17, and MUC-13, or a functional fragment or variant thereof.
  • the engineered WNT signal enhancing molecule comprises a first domain that binds to one or more E3 ubiquitin ligases, and a second domain that binds to a tissue specific receptor.
  • the E3 ubiquitin ligases are selected from the group consisting of Zinc and Ring Finger Protein 3 (ZNRF3) and Ring Finger Protein 43 (RNF43).
  • the first domain comprises an R-spondin (RSPO) polypeptide.
  • RSPO polypeptide is selected from the group consisting of RSPO-1, RSPO- 2, RSPO-3, and RSPO-4.
  • the RSPO polypeptide comprises a first furin domain and a second furin domain.
  • the second furin domain is wild-type or is mutated to have lower binding to Leucine-rich repeat-containing G protein coupled receptors 4-6 (LGR4-6).
  • LGR4-6 Leucine-rich repeat-containing G protein coupled receptors 4-6
  • the engineered WNT agonist is disclosed in Table 3.
  • the engineered WNT agonist and the engineered tissue specific WNT signal enhancing molecule are administered with a binding domain that specifically binds an inflammatory molecule.
  • the binding domain specific for the inflammatory molecule is an antagonist of the inflammatory molecule.
  • the antagonist of the inflammatory molecule is an antagonist of TNFa, IL-12, IL-12 and IL-23, or IL-23.
  • the gastrointestinal disease is inflammatory bowel disease.
  • the inflammatory bowel disease is selected from the group consisting of: Crohn’s disease (CD), CD with fistula formation, and ulcerative colitis (UC).
  • the present invention provides for a method of treating a subject suffering from a gastrointestinal disorder comprising administering to the subject, an engineered WNT agonist and an engineered tissue specific WNT signal enhancing combination molecule.
  • the combination molecule comprises: a) the engineered WNT agonist comprising one or more binding domains that bind to FZD5, FZD8, FZD1, FZD2, FZD7, FZD 5and 8, or FZD1, 2, and 7, and one or more binding domains that bind to LRP5, LRP6, or LRP5 and b) the engineered WNT signal enhancing molecule comprising a first domain that binds to one or more E3 ubiquitin ligases, and a second domain that binds to a tissue specific receptor.
  • the E3 ubiquitin ligases are selected from the group consisting of Zinc and Ring Finger Protein 3 (ZNRF3) and Ring Finger Protein 43 (RNF43).
  • the first domain comprises an R-spondin (RSPO) polypeptide.
  • the RSPO polypeptide is selected from the group consisting of RSPO-1, RSPO-2, RSPO-3, and RSPO-4.
  • the RSPO polypeptide comprises a first furin domain and a second furin domain.
  • the second furin domain is wild-type or is mutated to have lower binding to Leucine-rich repeat-containing G protein coupled receptors 4-6 (LGR4-6).
  • combination molecule incorporates a tissue targeting molecule.
  • the tissue targeting molecule is an antibody or fragment thereof that binds to a tissue specific cell surface antigen.
  • the tissue targeting molecule is selected from the group consisting of GPA33, CDH17, and MUC-13, or a functional fragment or variant thereof.
  • the combination molecule is administered with a binding domain that specifically binds an inflammatory molecule.
  • the binding domain specific for the inflammatory molecule is an antagonist of the inflammatory molecule.
  • the antagonist of the inflammatory molecule is an antagonist of TNFa, IL-12, IL-12 and IL-23, or IL-23.
  • the gastrointestinal disease is inflammatory bowel disease.
  • the inflammatory bowel disease is selected from the group consisting of: Crohn’s disease (CD), CD with fistula formation, and ulcerative colitis (UC).
  • the engineered WNT agonist is selected from those disclosed in any of the following: PCT Application Publication No. WO 2016/040895; US Application Publication No. US 2017-0306029; US Application Publication No. US 2017-0349659; PCT Application Publication No. WO 2019/126398; or PCT Application Publication No. WO 2020/01030.
  • the tissue-specific WNT signal enhancing molecule is selected from those disclosed in any of the following: PCT Application Publication No. WO 2018/140821; US Application Publication No US 2020-0048324; or PCT Application Publication No. WO 2020/14271, all of which are herein incorporated by reference in their entireties.
  • the disclosure provides a method of treating a subject suffering from a gastrointestinal disorder comprising administering to the subject an engineered WNT agonist, an engineered WNT signal enhancing molecule, and/or a combination molecule disclosed herein, or a pharmaceutical composition comprising an engineered WNT agonist or combination molecule disclosed herein.
  • the gastrointestinal disorder is an inflammatory bowel disease, optionally selected from the group consisting of: Crohn’s disease (CD), CD with fistula formation, and ulcerative colitis (UC). Any of the methods disclosed herein may be practiced using any of the engineered WNT agonists, engineered WNT signal enhancing molecules, and/or combination molecules disclosed herein.
  • the WNT agonist is administered to a subject, e.g., a mammal, intravenously, e.g., as a bolus injection.
  • the WNT agonist is administered at least once per week.
  • the subject is administered about 0.5 to about 100 mg/kg body weight of the WNT agonist, or about 2 to about 50 mg/kg body weight of the WNT agonist, e.g., about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg.
  • the subject is administered about 3 to about 30 mg/kg body weight intravenously at least once per week of R2M13-h26, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO:9 and two polypeptides of SEQ ID NO: 10 bound by disulfide bonds.
  • the method is used to treat IBD, e.g., moderate to severe IBD with a WNT agonist disclosed herein, e.g., R2M13-h26.
  • the IBD Crohn’s disease, Crohn’s disease with fistula formation, or ulcerative colitis are examples of IBD Crohn’s disease, Crohn’s disease with fistula formation, or ulcerative colitis.
  • any of the methods disclosed herein may also be practiced using a combination of a WNT agonist molecule and a tissue-specific WNT signal enhancing molecule or a combination molecule comprising both a WNT agonist molecule and a tissue-specific WNT signal enhancing (combination molecule), e.g., as described herein.
  • a WNT agonist molecule and/or a tissue-specific WNT signal enhancing molecule, or combination molecule is provided to a subject having a disease involving inappropriate or deregulated WNT signaling.
  • methods disclosed herein comprise providing to a subject in need thereof a WNT agonist molecule and/or a tissue-specific WNT signal enhancing molecule, alone or in combination, or a combination molecule.
  • a WNT agonist molecule and a tissue-specific WNT signal enhancing molecule are provided to the subject in the same or different pharmaceutical compositions.
  • the WNT agonist molecule and the tissue-specific WNT signal enhancing molecule are provided to the subject at the same time or at different times, e.g., either one before or after the other.
  • the methods comprise providing to the subject an effective amount of a WNT agonist molecule and/or tissue-specific WNT signal enhancing molecule.
  • an effective amount of the WNT agonist molecule and the tissue-specific WNT signal enhancing molecule are present in the subject during an overlapping time period, e.g., one day, two days, or one week.
  • methods disclosed herein comprise providing to a subject in need thereof a combination molecule comprising a WNT agonist molecule and a tissue-specific WNT signal enhancing molecule (combination molecule).
  • any of the methods disclosed herein may be practiced to reduce inflammation (e.g., inflammation associated with IBD or in a tissue affected by IBD, such as gastrointenstinal tract tissue, e.g., small intestine, large intestine, or colon), increase WNT signaling, reduce any of the histological symptoms of IBD (e.g., those disclosed herein), reduce cytokine levels in inflamed tissue (e.g., gastrointenstinal tract tissue), or reduce disease activity index as disclosed herein.
  • inflammation e.g., inflammation associated with IBD or in a tissue affected by IBD, such as gastrointenstinal tract tissue, e.g., small intestine, large intestine, or colon
  • increase WNT signaling reduce any of the histological symptoms of IBD (e.g., those disclosed herein)
  • reduce cytokine levels in inflamed tissue e.g., gastrointenstinal tract tissue
  • reduce disease activity index as disclosed herein.
  • a WNT agonist molecule or tissue-specific WNT signal enhancing molecule or combination molecule may be used to enhance a WNT signaling pathway in a tissue or a cell.
  • Agonizing the WNT signaling pathway may include, for example, increasing WNT signaling or enhancing WNT signaling in a tissue or cell.
  • the present disclosure provides a method for agonizing a WNT signaling pathway in a cell, comprising contacting the tissue or cell with an effective amount of a WNT agonist molecule and/or a tissue-specific WNT signal enhancing molecule, or a combination molecule, or pharmaceutically acceptable salt thereof, disclosed herein, wherein the WNT agonist molecule and/or tissue-specific WNT signal enhancing molecule, or combination molecule is a WNT signaling pathway agonist.
  • the disclosure provides a method of increasing WNT signaling in a cell, comprising contacting the cell with an engineered WNT agonist disclosed herein.
  • the WNT agonist is R2M13-h26.
  • contacting occurs in vitro, ex vivo, or in vivo.
  • the cell is a cultured cell, and the contacting occurs in vitro.
  • the WNT agonist and/or tissue-specific WNT signal enhancing molecule, or combination molecule may be used for the treatment of gastrointestinal disorders, including but not limited to, inflammatory bowel disease, including but not limited to, Crohn’s disease, Crohn’s disease with fistula formation, and ulcerative colitis.
  • the WNT agonist may be used for the treatment of gastrointestinal disorders, including but not limited to, inflammatory bowel disease, including but not limited to, Crohn’s disease with or without fistula formation, including but not limited to ulcerative colitis, including but not limited to acute intestinal GVHD (Graft versus host disease), including but not limited to Short Bowel Syndrome and any other gastro-intestinal disease where the epithelial barrier is impaired or the intestine is shortened.
  • the present invention provides a WNT/ b-catenin signaling WNT/[3-catcnin agonist to enhance regeneration of the intestinal epithelium as a result of injury from these disorders.
  • the WNT agonist is R2M13- h26.
  • the engineered WNT agonists may also be used to modulate a variety of tissue and/or cellular process, and to modulate gene expression within tissues and/or cell.
  • the disclosure provides methods of modulating gene expression, comprising contact a subject, organ, tissue, or cells with an engineered WNT agonist disclosed herein, e.g ., in Table 3.
  • the subject may be administered the engineered WNT agonist, and the organ, tissue, or cells may be contacted with the engineered WNT agonist in vivo, ex vivo, or in vitro.
  • the method results in upregulation or downregulation of one or more genes in the WNT signaling pathway, including but not limited to any of the genes disclosed in Tables 4-8.
  • Upregulation or downregulation of gene expression may be measured at the RNA or protein level, and may result in an increase of at least two-fold, at least five-fold, at least 10-fold, or at least 20-fold or a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in one or more tissues and/or cells of the subject following administration.
  • the increase or decrease may be determined based on comparison to a pre-determined control level or the level determined for corresponding cells or tissue not contacted with the engineered WNT agonist, in certain embodiments.
  • the disclosure provides a method of modulating expression of a WNT pathway molecule in one or more tissues and/or cells in a subject having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or the pharmaceutical composition disclosed herein.
  • the WNT pathway molecule is a gene or protein listed in any one of Tables 4-7.
  • the WNT pathway molecule is selected from the group consisting of: RNAse4, Angiongenin, Gsta3, Rnf43, Axin2, Ccnbl, or any of the genes or proteins listed in Table 7.
  • expression of the WNT pathway molecule is increased by at least 1.1-fold, at least 1 2-fold, at least 1 3 -fold, at least 1.4-fold, at least 1.5-fold, at least two-fold, at least five-fold, at least 10-fold, or at least 20-fold or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in one or more tissues and/or cells of the subject following administration of the engineered Wnt agonist.
  • the tissue is epithelial tissue.
  • the cells are gastrointestinal epithelial cells, optionally: stem cells, TA1, TA2, basal goblet, injury -induced alternative progenitors (AltEnteroPC), injury-induced alternative enterocytes (Alt Entero), enterocyte precursors (EnteroPrecur), goblet cells 1, goblet cells 2, enteroendocrine or tuft cells.
  • the WNT agonist is R2M13-h26.
  • the disclosure provides a method of stimulating tissue repair in a subject having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or the pharmaceutical composition disclosed herein.
  • the tissue repair is stimulated by (or the method results in) modulation of at least one WNT pathway molecule selected from the group consisting of: genes associated with the cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and/or any of the genes listed in any of Tables 4-8.
  • the genes associated with the cell cycle are selected from those provided in Table 4, or Aurka, Aurkb, Ccna2, Ccnbl, Ccnb2, Ccnd2, Ccnel, Cdc45, Cdkl, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plkl, Rrml, and Rrm2.
  • the genes associated with stem and progenitor cell renewal and differentiation are selected from those provided in Table 8, and Axin2, Idl, Hmga2, Nhp2, Foxql, and Adhl.
  • the genes associated with epithelial cell repair and barrier restoration are selected from those provided in Table 6, or Apexl, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zgl6, and Sprr2a3.
  • expression of the gene is increased by at least two-fold, at least five-fold, at least 10-fold, or at least 20- fold or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in one or more tissues and/or cells of the subject following administration of the engineered Wnt agonist.
  • the WNT agonist is R2M13-h26.
  • the disclosure provides a method of reducing inflammation in a subject having a gastrointestinal disorder (or a tissue or cells thereof), comprising administering to the subject an engineered WNT agonist or the pharmaceutical composition disclosed herein.
  • the inflammation is reduced by (or the method results in) modulation of at least one WNT pathway molecule selected from the group consisting of: genes provided in Table 5, or Adamdecl, Atf3, Gpx2, Gsta3, Gstml, Gdfl5, 1118, Noxl, Reg4, Sycn, Selenbpl, Tgfbr2, and Timp3.
  • the inflammation is reduced in gastrointestinal tissue, optionally epithelial tissue.
  • the inflammation is reduced in gastrointestinal epithelial cells, epithelial stem cells, TA1, TA2, basal goblet cells, injury-induced alternative progenitors (AltEnteroPC), injury-induced alternative enterocytes (AltEnteros), enterocyte precursors (EnteroPrecur), goblet cells 1, goblet cells 2, or enteroendocrine cells.
  • AltEnteroPC injury-induced alternative progenitors
  • AltEnteros injury-induced alternative enterocytes
  • enterocyte precursors EnteroPrecur
  • goblet cells 1 goblet cells 2, or enteroendocrine cells.
  • expression of the WNT pathway molecule is increased by at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5- fold, two-fold, at least five-fold, at least 10-fold, or at least 20-fold or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in one or more tissues and/or cells of the subject following administration of the engineered Wnt agonist.
  • the WNT agonist is R2M13-h26.
  • the WNT agonist molecule may also incorporate a tissue targeting moiety, e.g., an antibody or fragment thereof that recognizes a pulmonary tissue specific receptor or cell surface molecule.
  • a tissue targeting moiety e.g., an antibody or fragment thereof that recognizes a pulmonary tissue specific receptor or cell surface molecule.
  • the present invention also provides for combination treatment with known and new treatments for gastrointestinal disorders, in particular inflammatory bowel diseases (IBD).
  • IBD inflammatory bowel diseases
  • the WNT agonist can be combined with several known therapies for IBD, including, but not limited to, 5-Aminosalicylates (5-ASAs); immunosuppressants such as corticosteroids, azathioprine or 6-mercaptopurine, methotrexate, and ciclosporin-A or tacrolimus; TNFa inhibitors such as infliximab, adalimumab, and golimumab; anti-integrins such as vedolizumab; inflammatory cytokine antagonists such as ustekinumab; janus kinase (JAK) inhibitors such as tofacitinib; SMAD 7 inhibitors such as mongersen; and SIP modulators, such as ozanimod and etrasimod; and any new agents that may come on the market for the
  • the therapeutic agent e.g., an engineered WNT agonist and/or tissue-specific WNT signal enhancing molecule or combination molecule
  • the subj ect therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • the subject method results in a therapeutic benefit, e.g., preventing the development of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc.
  • the subject method comprises the step of detecting that a therapeutic benefit has been achieved. The ordinarily skilled artisan will appreciate that such measures of therapeutic efficacy will be applicable to the particular disease being modified, and will recognize the appropriate detection methods to use to measure therapeutic efficacy.
  • Fluorescent reagents suitable for modifying nucleic acids including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available.
  • Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.
  • RNAscope in situ hybridization ACD Bio.
  • RNAscope probes used are listed below.
  • standard RNAscope® 2.5 HD Assay-Red protocol www.acdbio.com
  • images were acquired on a Leica DMi8 microscope equipped with a DFC7000T color camera.
  • fluorescent RNAscope in situ hybridization standard RNAscope Multiplex Fluorescent Reagent Kit v2 Assay protocol was followed (ACD Bio Document #323100-USM) and coupled with the TSA Plus Cyanine 3 and 5 Systems. Fluorescent images were acquired with a Leica Thunder imaging system.
  • MagMAXTM mirVana (Thermofisher, A27828) Total RNA Isolation Kit was used for RNA isolation on a KingFisher (Thermofisher) sample purification system. Reverse Transcription was done with the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Thermofisher, 4368814), and the Applied Biosystems TaqMan Fast Advanced Master Mix (Thermofisher, 4444557) was used for qPCR.
  • Binding kinetics of R2M13, the Fzd binding portion of R2M13-26, Fab to each CRD of Fzd5,8 was determined by bio-layer interferometry (BLI) using Octet Red 96 (PALL ForteBio, Fremont, CA) instrument at 30°C, 1000 rpm with Streptavidin (SA) biosensors. Biotinylated CRDs of Fzds diluted to 25 nM in the running buffer (PBS, 0.05% Tween-20, 0.5% BSA, pH 7.2) were captured to the SA biosensor followed by dipping into wells containing the R2M13 Fab protein at different concentrations in running buffer or into a well with only running buffer as a reference channel.
  • BSA Streptavidin
  • Basal Media+IWP2+anti- ⁇ Gal or Basal Media+IWP2+Wnt mimetic was applied to the wells. Each condition included 5-6 repeats. Media and treatments were changed once on Day 4 after plating. Images of the 3D cultured organoids were acquired on Day 7.
  • mice Seven-week-old C57B1/6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were housed 4-5 per cage. All animal experimentation was in accordance with the criteria of the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences Protocols for animal experimentation were approved by the Surrozen Institutional Animal Care and Use Committee. Mice were acclimatized a minimum of two days prior to initiating experiments. Mice were kept 12/12- hour light/dark cycle in a 30% to 70% humidity environment and room temperature ranging from 20°C to 26°C.
  • DSS Sodium (DSS, MP Biomedicals, molecular weight 36-50kDa, Ref# 160110) in drinking water from day 1 to day 7 to induce colitis and were switched to 1% DSS from day 8. Protein treatments were dosed either once on day 7 or twice on day 4 and 7. Animals were terminated on day 10, allowing a 6-day course of protein treatment, and the colon was harvested for histology and RT-qPCR. In one of the studies, DSS induced mouse body weight loss for animals treated with anti-GFP was nearly 25% on day 9 so animals were switched to drinking water with no DSS for compliance with IACUC rules.
  • the disease activity index was calculated based on the average score of weight loss, stool consistency and the degree of intestinal bleeding (Wirtz Stefan et al., 2017) Scoring system by grading was on a scale of 0 to 4 using the following parameters: loss of weight (0, 0-1%; 1, 1-6%; 2, 6-12%; 3, 12-18%; 4, >18%), stool consistency (0, normal; 1, soft but still formed; 2, soft; 3, very soft, wet; 4, watery diarrhea) and intestine bleeding (0-1, negative hemoccult; 2, positive hemoccult; 3, blood traces in stool visible; 4, gross rectal bleeding).
  • Small intestine and colon were extracted and, after removing fecal content, weighted and length measured.
  • the desired small intestine segments (duodenum, jejunum, ileum) and colon segments (ascending, transverse and descending colon) were cut out and fixed directly in 10% neutral buffered formalin (NBF) overnight.
  • NBF neutral buffered formalin
  • Tissues were then transferred to 70% ethanol before paraffin embedding. Paraffin tissue blocks were then sectioned to 5 mM thickness and stained with hematoxylin and eosin (H&E) for histology analysis. Pathology reading was performed by an independent pathologist.
  • H&E hematoxylin and eosin
  • tissue sections on slides were deparaffinized followed by citrate buffer (pH 6) antigen retrieval in a steamer. Slides were then washed thoroughly in tap water followed by lx wash in PBST. Subsequently, tissue sections were blocked with serum free protein block (Agilent, X090930-2) for one hour at room temperature followed by incubation in primary antibodies. After primary antibody incubation, tissue sections were washed in 0.1 % TX-100 in PBS (PBST) at least three times followed by incubation in secondary antibody. Afterwards, tissue sections were washed with PBST, and coverslips were mounted with Vectashield Vibrance antifade mounting medium with DAPI (Vector Laboratories, H-1800).
  • DAPI Vectashield Vibrance antifade mounting medium
  • Mouse colon was dissociated as described below and resuspended in FACS buffer (HBSS, 2% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 1 % Pen-strep or antibiotic/antimycotic solution). Prior to FACS, cells were passed through a 40-micron filter, and DAPI was added to distinguish live/dead cells. Prior to target antibody incubation, FcR blocking reagent (Miltenyi Biotec, 130-092-575) was added to the samples and incubated for 10 minutes.
  • FACS buffer HBSS, 2% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 1 % Pen-strep or antibiotic/antimycotic solution.
  • FcR blocking reagent Miltenyi Biotec, 130-092-575
  • RNA-sequencing seq Single cell RNA-sequencing seq : tissue dissociation, cell isolation, library preparation, sequencing
  • mice were treated with 4% DSS in their drinking water throughout the duration of the experiment.
  • DSS-treated animals were dosed with 10 mpk R2M13-26 or an anti-GFP antibody on day 4 of the DSS treatment.
  • Cells from two uninjured, naive mice (no DSS) at day 5 and day 6 and from three replicates each for anti-GFP and R2M13-26-treated DSS animals were collected for each timepoint. Each animal was considered a replicate.
  • Transverse colon was isolated from each animal and feces were removed After a brief wash in cold PBS, the colon was cut longitudinally to open the tube into a flat sheet, and the tissue was cut into 3-4 mm length fragments. Tissue fragments were incubated in prewarmed (37 °C) PBS with 5 mM EDTA in a shaker at 37 °C at 150 rpm for 15 minutes. After 15 minutes, the tubes containing the samples were vigorously shaken for 10 seconds to release more epithelial cells. The epithelial cells floating in suspension were removed to a new tube and centrifuged at 200 ref for two minutes
  • the residual tissue containing the remaining epithelia and stroma/lamina intestinal was then incubated in 8-12.5 mL of lamina propria dissociation buffer (AdvDMEM/F12 with 10 mM HEPES, 0.2 % FBS, DNAse 1 (80 U/mL), Liberase TM (0.2 mg/mL), and 1 % antibiotic/antimycotic) at 37 °C for 30 minutes with horizontal shaking at 150 rpm.
  • the epithelial cells were resuspended in 1 mL of TiypLE with DNasel, and they were incubated at 37 °C for five minutes and triturated with a P1000 pipette for 30 seconds.
  • a shared nearest neighbor (SNN) graph-based clustering method (Xu & Su, 2015) was applied by using the wrapper function (buildSNNGraph) from the R package scran (version 1.18.5) with k equal to 40 coupled with the cluster louvain function from the R package igraph (1.2.6) to the first 10 principal components derived from the top 2000 most variable genes across the data set.
  • the data were subsetted into these three smaller data sets, and the cells within each layer/lineage were clustered using the SNN graph-based method and the walktrap algorithm implemented with the cluster walktrap function from the igraph package, applied to the first 15 principal components derived from the top 2000 most variable genes within that subsetted layer/lineage (immune, stromal, epithelial). Cell type/subtype identities were determined using established marker genes and published literature.
  • GSEA Gene set enrichment analysis
  • R package edgeR version 3.32.1
  • MSigDB Molecular Signature Database
  • C2 Hallmark and curated gene sets of the KEGG, Biocarta, PID, Reactome, ST, SIG, an SA types.
  • MSigDB Molecular Signature Database
  • C2 Hallmark and curated gene sets of the KEGG, Biocarta, PID, Reactome, ST, SIG, an SA types.
  • the kegga function of the edgeR package was also implemented, which only uses KEGG pathways, and similar results were observed (data not shown).
  • GSEA was applied in both pairwise and more specific contrasts to the pseudobulk samples aggregated by replicate.
  • Engineered Wnt agonists in the IgGl format were synthesized, including Wnt agonists having humanized Lrp5/6 binding domains fused to the N-terminus of each light chain of a Fzd binding antibody.
  • An illustrative structure is shown in Figure 1.
  • the Lrp5/6 binding domain was derived from various camelid single chain antibody (VHH) binding domains selected from: VHH03, VHH26, or VHH36.
  • the VHH03 domain binds Lrp5; the VHH26 domain binds Lrp6; and the VHH36 domain binds Lrp5 and Lrp6.
  • the camelid single chain antibody was humanized by retaining the CDR sequences but replacing other sequences with a human antibody backbone.
  • the resulting LRP5/6 binding domain was modified to remove potential liabilities.
  • VHH26 Humanization of VHH26 was performed as described below. Humanization of camelid VHH domains is considered to be challenging as they are derived from single-chain, homodimeric antibodies lacking VL:CL or VH:CH interactions present in hetrotetrameric human IgGl antibodies. Surface properties of camelid VHHs (Muyldermans (2Q ⁇ 3)Annu. Rev. Biochem 82:775-797; Vincke et al (2009) J. Biol. Chem. 284: 3273-3284) are evolutionarily reshaped to optimize the stability of homodimeric nature of single-chain antibodies. Humanization of camelid VHH26 was performed initially by CDR-grafting (for review: Safdari et al., (2013) Biotechnol.
  • VHH26 variants HI to H6 and the parental VHH26 were transiently expressed in Expi293 cells (at 80mL scale) with a C-terminal hexa-histidine tag. Proteins were purified using His-Complete resin (Roche, USA) following standard procedures. Expression levels and homogeneity of VHH26 and its humanized variants were analyzed by SDS-PAGE and SEC (size-exclusion chromatography).
  • binding kinetics of VHH26-H1, VHH26-H2, VHH26-H3, VHH26-H4, VHH26-H5, VHH26-H6, and VHH26 His to biotinylated LRP6E3E4 were determined by bio-layer interferometry (BLI) using Octet Red 96 (PALL ForteBio, Fremont, CA) instrument at 30°C, 1000 rpm Streptavidin (SA) biosensors.
  • Biotinylated LRP6E3E4 diluted to 50 nM in the running buffer (PBS, 0.05% Tween-20, 0.5% BSA, pEl 72) were captured to the SA biosensor followed by dipping into wells containing the indicated VHH26 proteins at different concentrations in running buffer or into a well with only running buffer as a reference channel.
  • KD for each binder was calculated by Octet System software, based on fitting to a 1 : 1 binding model.
  • Kinetics values (Kon, Koff, KD) from each experiment was calculated from seven technical replicates of different concentrations of the molecule tested with Octet Data Analysis 9.0 (PALL ForteBio, Fremont, CA).
  • VHH26-H5 was used in the further experiments as a humanized LRP binding domain fused to a Fzd binding domain, e.g., tetravalent, bispecific WNT agonists.
  • the Fzd binding domain was derived from the R2M13 antibody, which binds Fzd5 and Fzd8, and included an effector-less Fc region that retained FcRn binding, e.g., LALAPG or N297G (Wang X et ah, Protein Cell 2018, 9:63-73)
  • N297G is an aglycosylated form of IgGl antibody, in which Asn is substituted by Gly.
  • N297 corresponds to amino acid N302, so the N297G mutation may alternatively be referred to as N302G.
  • LALAPG represents three mutations in the Fc domain of IgGl. Using a canonical IgGl sequence numbering, Leu234 and Leu235 were mutated to Ala; similarly, Pro329 was mutated to Gly. Hence this triple-mutant in the Fc domain is referred to as “LALAPG”. In the case of R2M13-h26, these mutations are in sequence positions, 239, 240, and 334, respectively.
  • VHH26-H5 was fused to the N-terminus of the light chains of the R2M13 antibody via a five amino acid linker, thus producing an IgG-like molecule comprising the R2M13 antibody with the VHH at the N-terminus of both antibody light chains.
  • Sequences of the R2M13 heavy chain IgG and the R2M13 light chain fused to the various LRP5/6 VHH binding domains by an amino acid linker present in the various Wnt agonists are provided in Table 3.
  • the sequences of the heavy and light chains present in the parental R2M13-03, R2M13-26, R2M13-36 Wnt agonists without LALPG or N297G modifications are provided as SEQ ID NOs: 136-138 (light chains, respectively) and SEQ ID NO: 153 (heavy chains) in PCT Application Publication No. WO2019/126398, which is incorporated herein by reference in its entirety.
  • the indicated Wnt agonist includes two of the heavy chains and two of the light chains in an antibody-type format, where the chains are connected via disulfide bonds.
  • R2M13-humanized_26-LALAPG construct (“R2M13-26 humanized LALAPG”, also referred to herein as R2M13-h26, R2M13-h26- LALAPG, or humanized LALPG) showed the highest activity of the humanized Lrp binding domains.
  • the R2M13-humanized_26-N297G construct (R2M13-26 humanized N297G; humanized N297G) was not stable. Humanization of VHH03 and VHH36, when paired with R2M13, reduced in vitro potency significantly, although their absolute EC50 values were comparable to VHH26 paired with R2M13.
  • R2M13-humanized_26-LALAPG construct The sequences of the heavy and light chains of the R2M13-humanized_26-LALAPG construct (R2M13-h26) are shown in Figure 6.
  • the construct comprised two heavy chains and two light chains bound by disulfide bonds.
  • the LALAPG mutations in the Fc domain removed effector function (see, e.g., Wang, et al. (2016) Protein Cell. 9:63-73).
  • the various domains of the R2M13-h26 construct are shown, and the domains of the other constructs can be readily determined based on these.
  • mice To induce acute colitis, 7- to 8-week-old female mice were given drinking water containing 4.0% (w/v) Dextran Sulfate Sodium (DSS, MP Biomedicals, MFCD00081551) ad libitum for 7 days followed by drinking water containing 1.0% (w/v) DSS for 3 days. Groups of mice were either untreated, treated with an isotype control antibody (anti-GFP), or treated with the indicated engineered Wnt agonist once on day 4 or twice on day 4 and 7.
  • anti-GFP isotype control antibody
  • DAI disease activity index
  • Various engineered humanized Wnt agonists were tested in the DSS model of acute colitis as outlined in Figure 7.
  • the constructs tested included non-humanized and humanized versions, including: R2M 13 -03 -LALAPG, R2M13-26-LALAPG, R2M13-36-LALAPG, R2M 13 -humanized-03 -LALAPG, R2M13-humanized-26-LAL APG, R2M 13 -humanized-36- LALAPG, R2M13 -humanized-03 -N297 G, and R2M13-humanized-36-N297G.
  • mice Six-week old C57B1/6J female mice (total of 96) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were housed 5 per cage. All animal experimentation was in accordance with the criteria of the ‘ ‘ Guide for the Care and Use of Laboratory Animals’ ’ prepared by the National Academy of Sciences Protocols for animal experimentation were approved by the Surrozen Institutional Animal Care and Use Committee. Mice were acclimatized a minimum of two days prior to initiating experiments. Mice were kept 12/12- hour light/dark cycle in a 30% to 70% humidity environment and room temperature ranging from 20°C to 26°C.
  • mice To induce acute colitis, 7- to 8-week-old female mice were given drinking water containing 4.0% (w/v) Dextran Sulfate Sodium (DSS, MP Biomedicals, MFCD00081551) ad libitum for 7 days followed by drinking water containing 1.0% (w/v) DSS for additional 3 days (Figure 7). Groups of mice were either untreated, treated with an isotype control antibody (anti-GFP), or treated with 1 mg per kg of the indicated engineered Wnt agonist on day 4 and day 7. All protein treatments showed comparable serum antibody exposure at termination
  • Control-treated animals subjected to DSS developed severe colitis characterized by profound and sustained weight loss and bloody diarrhea, resulting in the increase of disease activity index as represented by fecal score.
  • Treatment with humanized R2M13-26 and humanized R2M13-36, either in the LALAPG or in the N297G form significantly improved body weight in DSS mice.
  • These constmcts also significantly decreased Disease Activity Index (DAI) in the DSS mice ( Figure 8); decreased fecal score in DSS mice; increased colon length and feces in DSS mice; and increased colon length and weight in DSS mice.
  • DAI Disease Activity Index
  • humanized R2M13-26 H-LALAPG 26
  • humanized R2M13-36 H-LALAPG 36
  • TNF-a tumor necrosis factor alpha
  • IL-6 IL-6
  • IL-8 interleukin-8
  • lipocalin-2 lipocalin-2
  • R2M13-26-LALAPG R2M13- h26-LALAPG
  • R2M13- h26-LALAPG humanized R2M13-26-LALAPG
  • Figure 11 humanized R2M13-26-LALAPG
  • Figure 12 repair damaged colon epithelium
  • Figure 13 restore the epithelial cell lineage including colonocytes, goblet cells, and tuft cells
  • Pharmacokinetics 1PK] of Engineered Wnt Agonists [00322] Pharmacokinetics (PK) of the parental R2M13-26 (R2M13-26-LALAPG) and humanized R2M13-26 (R2M13-h26-LALAPG) following intravenous injection was determined by measuring the amount of antibody in serum at various times following administration to rats and compared to data obtained from mice ( Figure 14). Cmax for humanized R2M13-26 (R2M13-h26) was higher than for parental R2M13-26 (R2M13-26), so differences carry over time; however, the fold difference increased over time.
  • Humanized R2M13-26 had lower clearance (25.3 mL/day/kg) than parental R2M13-26 (40.0mL/day/kg), and humanized R2M13-26 had a longer half-life (3.75 days) than parental R2M13-26 (2.47 days).
  • Example 6 Humanized R2M13-26 had lower clearance (25.3 mL/day/kg) than parental R2M13-26 (40.0mL/day/kg), and humanized R2M13-26 had a longer half-life (3.75 days) than parental R2M13-26 (2.47 days).
  • mice Six-eight-week old C57B1/6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were housed 4-5 per cage. All animal experimentation was in accordance with the criteria of the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences Protocols for animal experimentation were approved by the Surrozen Institutional Animal Care and Use Committee.
  • mice were given three cycles of drinking water containing 3.0% (w/v) Dextran Sulfate Sodium (DSS, MP Biomedicals, MFCD00081551) ad libitum for 5 days followed by plain drinking water for 7 days. Groups of mice were either treated with an isotype control antibody (anti-GFP), or treated with 4 doses of R2M13-26- LALAPG (R2M13-26) at 10 mg/kg on days 16, 19, 28 and 31. Animals were terminated on day 33
  • R2M13-26 treatment improved body weight and disease activity index in the chronic DSS model.
  • R2M13-26 also improved colon histology.
  • R2M13-26 reduced the serum inflammatory mediators IL-6 and lipocalin-2 on day 33 at termination of the study (data not shown).
  • Examples 3 and 4 demonstrated that the Fzd5,8 specific R2M13-26 and R2M13- h26 Wnt agonists were effective in treating acute mouse colitis (acute DSS) model.
  • the goal of this study was to develop a more comprehensive understanding of the mechanism of action by which R2M13-26 affects cells in the colon throughout the repair process using a similar model system.
  • mice were either untreated, treated with a control antibody (anti-GFP), or treated with a single i.p. injection of R2M13-26-LALAPG (R2M13-26) on day 4.
  • Daily food intake, BW, fecal score, and occult blood were measured.
  • RT-qPCR analysis was performed on bulk colon samples to evaluate changes in gene expression.
  • Examination of Wnt induction showed a significant decrease in Axin2 with DSS.
  • R2M13-26 induced expression of Axin2 under no DSS condition.
  • Examination of proliferation markers showed a significant decrease in Ki67 with DSS on Day 4 & rescue by R2M13-26.
  • R2M13-26 rescued Cdkl downregulation in the presence of DSS.
  • Analysis of stem cell markers showed Lrigl significantly decreased with DSS on Day 4 and was rescued by R2M13-h26. With respect to clinical markers for IBD, significant upregulation of Gpx2 was seen on Days 5 & 6.
  • mice were treated with 4% DSS in their drinking water throughout the duration of the experiment.
  • DSS-treated animals were dosed with 10 mpk R2M13-26 or an anti-GFP antibody on day 4 of the DSS treatment.
  • On day 5 and day 6, three each of Wnt agonist and anti-GFP dosed animals were collected at what was 24-hours and 48-hours post dosing, respectively.
  • Two naive, uninjured animal samples were also collected at the day 5 and 6 timepoints. Colon, small intestine, spleen and liver tissues were collected at termination and examined or frozen for mRNA analysis.
  • Single cell RNA sequencing (scRNA-seq) was performed on fresh transverse colon samples for single cell isolation, and RT-qPCR was performed on fresh transverse colon for isolation of epithelium only.
  • Transverse colon was isolated from each animal and feces were removed After a brief wash in cold PBS, the colon was cut longitudinally to open the tube into a flat sheet, and the tissue was cut into 3-4 mm length fragments. Tissue fragments were incubated in prewarmed (37 °C) PBS with 5 mM EDTA in a shaker at 37 °C at 150 rpm for 15 minutes. After 15 minutes, the tubes containing the samples were vigorously shaken for 10 seconds to release more epithelial cells.
  • the epithelial cells floating in suspension were removed to a new tube and centrifuged at 200 ref for two minutes to pellet the epithelial cells that dissociated from the tissue
  • the residual tissue containing the remaining epithelia and lamina intestinal was then incubated in 8-12.5 mL of lamina propria dissociation buffer at 37 °C for 30 minutes with horizontal shaking at 150 rpm.
  • the epithelial cells were resuspended in 1 mL of TrypLE with DNasel, and the epithelial cells were incubated at 37 °C for about eight minutes and triturated with a P 1000 pipette about 25 times.
  • the epithelial cells were centrifuged at 500 ref, 4 °C, and the supernatant was removed. Epithelial cells were then washed one time in FACS buffer before another round of centrifugation and final resuspension in 0.5 mL of FACS buffer. Following 30 minutes of dissociation in LP dissociation buffer, the remaining tissue fragments and suspension were centrifuged at 500 ref for five minutes. Supernatant was removed down to 1 mL, and the sample was triturated with a PI 000 until the solution was homogeneous and all tissue fragments had dissociated. After trituration, the sample was centrifuged at 500 ref for 5 minutes at 4 °C and washed in FACS buffer prior to being resuspended in 1 mL of FACS buffer in preparation for FACS.
  • Illumina read data was processed using the lOx Genomics Cellranger pipeline. Demultiplexed UMI count data was then assessed and low-quality cells and low expression genes were removed. UMI count data was normalized using deconvolution scaling from the R package scran , and cells were clustered using a SNN graph-based clustering approach using the R package scran. Cell type identities were determined using established cell type markers. Differential gene expression was performed at the single cell level for each cluster at using one versus all and pair-wise comparisons within each lineage by using the R package clusterExperiment to run EdgeR.
  • Differential gene expression analysis between experimental conditions was performed with the R package edgeR on pseudobulk samples following aggregation of biological replicate samples based at the lineage level or at the cell type/cluster level. Differential expression comparisons were performed between experimental conditions (DSS-injured vems uninjured and then within the DSS-injury samples for R2M13-26-treated versus anti-GFP treated) along the epithelial lineage and within individual clusters representing cell types within the epithelial lineage for each timepoint (24-hours or 48-hours).
  • R2M13-26 exerted its effect predominately by directly impacting the epithelial cells of the colon due to the high expression of FZD5 on intestinal epithelial cells and its enrichment in the stem and progenitor cell populations.
  • the following Wnt target genes were increased when one compares the expression of the entire epithelial lineage and all of the cell types that it contains between R2M13-26 and control treatment (Table 7). Molecules were selected if they showed at least a two-fold increase between treatment and control across the epithelial lineage and they had been shown to be direct Wnt targets in the literature.
  • the majority of Wnt target genes were taken from the genetic manipulation and chromatin immunoprecipitation experiments published in Gougelet et al. (2014). Additional scRNA-seq data is shown in Tables 4-6, and 8.
  • the scRNA-seq data was used to examine specific cell types and compare gene expression between R2M13-26 treated cells and control treated cells to identify the Wnt target genes that are increased or decreased in each relevant cell type within the epithelial lineage.
  • a number of molecules were identified as significantly increased or decreased when compared to the expression of the aggregated epithelial lineage and/or any of the cell types that it contains between R2M13-26 and control treatment. Molecules were selected if they showed at least a two-fold change between treatment and control across the epithelial lineage or within at least one epithelial cell type in the acute DSS mouse model of IBD. These molecules are shown in Tables 4-8.
  • Wnt signaling is critical to maintaining and renewing the stem and progenitor cell pool and regulating their differentiation (Pinto et ah, 2003; Ma et ah, 2016).
  • R2M13-26 promoted repair and regeneration of the epithelium by maintaining the stem and progenitor cells, which was evidenced by increased expression of several key genes involved in this process (Table 8), including Idl (Hollnagel 1999; Meteoglu 2008; Ruzinova 2003), Nhp2 (Fong 2014; McCann 2020) and Hmga2 (Nishino 2008; Parisi 2020), Foxql (Tu 2018; Zhang 2018), and Aldhl (Tomita 2016). Furthermore, there was also an impact on expression of Areg, a ligand for EGFR signaling, which is important for intestinal stem cell niche maintenance (Fujii 2008; Mahtouk 2005; Suzuki 2010; Takahashi 2020).
  • glucagon can be processed into multiple small peptides, among them are GLP-1 and GLP-2, which play a role in reducing inflammation in IBD. GLP-2 also acts as a growth factor to promote stem and progenitor cell proliferation and regeneration of the epithelial crypts (Drucker 1999; Markovic 2019; Zatorski 2019). These data show that Wnt signaling activation increases expression of glucagon, which would lead to increased levels of GLP-2 and contribute to the expansion of the stem and progenitor cells.
  • R2M13- 26 promoted expression of many genes and pathways associated with reducing the inflammatory response in injury and IBD (Table 5). These molecules have an antiinflammatory effect and/or their reduction is associated with an increase in inflammation or worsening of IBD.
  • R2M13-26 treated groups showed dose response on serum antibody concentration at 24- and 48-hours post injection, and R2M13-26 exhibited linearity at 1, 3, and 10 mpk dosing
  • R2M13-26 increased Axin2 and Ki67 expression two days post-single I.P. injection ( Figure 25)
  • R2M13-26 increased LGR5 expression two days post-injection.
  • R2M13-26 treatment increased Occludin expression at 2 days post-injection.
  • Examples 3 and 4 demonstrated that the Fzd5,8 specific R2M13-26 and R2M13- h26 were effective in treating acute mouse colitis (acute DSS) model, and Example 6 demonstrated that R2M13-26 was effective in treating chronic mouse colitis (chronic DSS) model.
  • the goal of this study was to compare the effectiveness of R2M13-h26 in treating the colitis models to the effectiveness of other agents, including Cyclosporin A, anti-TNF antibodies, and anti-IL-12/23 antibodies.
  • Six to seven-week old C57B1/6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were housed 4-5 per cage. All animal experimentation was in accordance with the criteria of the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences. Protocols for animal experimentation were approved by the Surrozen Institutional Animal Care and Use Committee.
  • R2M13-h26 treatments improved body weight, decreased fecal score, and decreased Disease Activity Index (DAI) more than Cyclosporine A did ( Figure 16).
  • R2M13-h26 repaired colon epithelium in vivo more effectively than Cyclosporine A ( Figure 17), improved colon histology score (data not shown), and decreased serum levels of inflammatory cytokines more than Cyclosporin A (data not shown).
  • R2M13-h26 showed efficacy on repair of colon epithelium, improvement of histology and disease activity index (DAI), and reduction of inflammatory cytokines, at doses as low as 1 mg/kg twice a week or 2 mg/kg single dose. Cyclosporin A showed a mild effect on reducing DAI and lipocalin-2, and was in general much less effective than R2M13-h26.
  • R2M13-h26 decreased colon histology scores, improved body weight, decreased fecal score, and decreased DAI, whereas anti-TNF had no effect on these disease parameters ( Figure 20 and data not shown). R2M13-h26 also reduced serum inflammatory cytokine levels, lipocalin-2 and IL-6, more than anti-TNF in chronic in vivo model ( Figure 21).
  • Fzd5,8 specific R2M13-h26 at various dosing regimen was able to reach significant effects on repair of colon epithelium, improvement of histology and disease activity index, and reduction of inflammatory cytokines.
  • anti-TNF Ab failed to ameliorate epithelial damage or DAI in the chronic DSS mice.
  • R2M13-h26 treatment was administered at 0.1 and 1 mpk for 4 injections.
  • Anti-IL 12/23 administered at 3 or 10 mpk for 4 or 8 injections.
  • Anti-IL12/23p40 was clone C17.8, from Invivoplus Bioxcell. Readout was conducted on day 38.
  • R2M13-h26 treatments decreased Disease Activity Index (DAI) in the chronic DSS mouse model, while anti-IL12/23p40 treatments did not ( Figure 23).
  • R2M13-h26 treatments decreased serum cytokine levels more effectively than anti-IL12/23 ( Figure 24). This study confirmed that R2M13-h26 was able to repair damaged colon epithelium and decrease serum inflammatory cytokine levels in a chronic DSS mouse model, while the BioXcell’s anti-IL12/23p40 monoclonal antibody was not.
  • Fzd5 was shown to be highly expressed in the colon of a mouse model of colitis induced by dextran sodium sulfate, or DSS.
  • DSS exposure led to disruption of the intestinal barrier resulting in an inflammatory response similar to that seen in IBD patients.
  • R2M13-h26 was observed to bind to DSS-injured intestinal cells, stimulating Wnt signaling as measured by the expression of Axin2, a downstream target gene in the Wnt pathway, restoring tissue architecture, epithelial cell type composition and epithelial barrier function.
  • Mice exposed to DSS for seven days led to the breakdown of the intestinal barrier, which can be readily visualized in stained cross sections of the colon.
  • DSS Dextran Sodium Sulfite
  • RNAscope in situ hybridization analyses showed a reduction of mRNA expression of Wnt target genes Axin2, Lgr5, Rnf43 as well as Wnt ligands Wnt2b and Wnt5a in the colon epithelium and the surrounding mesenchymal cell layers, respectively.
  • the mRNA expression of the predominant mouse intestinal R-spondin, Rspo3 , in the mesenchymal cells underneath the colon crypts was not affected by DSS (see WO 2020/185960A1).
  • mice were injected with two doses of R2M3-26 or a Wnt mimetic targeting FZD1,2,5,7,8 and LRP6 referred to as FA-L6 in Fowler et al. (Fowler, T. W et al., (2021). Development of selective bispecific Wnt mimetics for bone loss and repair. Nature Communications, 72(1). https://doi.org/10.1038/s41467-021-23374-8), starting at day 4 when DSS damage to the epithelium was already visible followed by another dose at day 7, and its effect on epithelial repair was evaluated at day 10, a 6-day treatment.
  • the colon tissues were examined by a pathologist blinded to the treatment and scored for common colitis pathological features (see methods described in Example 1).
  • the histology score consistently showed R2M3-26 effectively repaired the DSS damaged colon tissue, reducing the colitis score from 4 75 to 2.0 (see WO 2020/185960A1).
  • RSPO alone or combined treatment of RSPO and the Wnt mimetic 18R5-DKKlc was previously shown to stimulate over proliferation of the small intestine stem cells and transient amplifying (TA) cells, leading to growth of small intestine crypt and villi length in normal mice (Yan Kelley S. et al., 2017).
  • TA transient amplifying
  • R2M13-26 a Fzd5,8-targeted Wnt mimetic stimulated growth of mouse intestinal organoids
  • RNAscope in situ hybridization analyses showed that, in the mouse small intestinal epithelium, Fzd5 was expressed at the highest level (Figure 29E), followed by Fzdl (Figure 29A) and Fzd7 (Figure 29G). Fzdl and Fzd7 were expressed mostly near the crypt bottom where Lgr5 positive stem cells reside ( Figure 29L). Expression of Fzd5 was concentrated near the crypt- villi border and in the crypt bottom columnar stem cells in the duodenum overlapping with the strong Axin2 positive domain, which was also positive for the stem cell marker Lgr5 (Figure 29K)
  • fecal score and disease activity index also improved with R2M13-26 and 1RC07-26 treatment (see WO 2020/185960A1). Improvement in fecal score and DAI was more pronounced with R2M13-26 as compared to R2M3-26 or 1RC07-26.
  • the colon tissue was again analyzed by a pathologist who was blinded to the treatment groups (see WO 2020/185960A1).
  • scRNA-seq was used to determine what cells first responded to treatment by R2M13-26, how R2M13-26 impacted differentiation of epithelial cells, and whether the effect on reducing inflammatory cytokines occurred directly on immune cells or indirectly through restoration of the epithelium. To study these questions, scRNA-seq was applied to investigate the early transcriptome response of the R2M13-26 treated colon in the acute DSS mouse model.
  • DSS DSS induced distinct cell types in each tissue layer or lineage, and this was responsible for a large portion of the lineage level differential gene expression. In the immune lineage, no cell types disappeared upon injury.
  • R2M13-26 promoted Wnt target and cell cycle gene expression and expanded the progenitor cell populations in the epithelium immediately following dosing.
  • R2M13-26 The direct effect of R2M13-26 was predominately on the epithelium. At a global level, at 24-hours after dosing, R2M13-26 led to the differential increase in expression of over 300 genes in the epithelium, but almost no or no genes in the immune and stromal cells/lineages (Figure 27). R2M13-26 increased expression of a wide range of Wnt target and cell cycle genes in the epithelium, both by expanding expression levels and by expanding the percentage of cells expressing the genes (Figure 27C; Tables 4 and 7).
  • Table 4 shows the cell cycle genes that were differentially expressed within the epithelial lineage when R2M13-26 treatment was compared to the anti-GFP treatment at either 24-hours or 48-hours Differential expression was filtered on adjusted p-value (false discovery rate (FDR)) of ⁇ 0.05.
  • R2M13-26 The predominant cell types impacted by R2M13-26 were the progenitor and precursor populations, including the injury-induced, altered enterocyte cell types. Differential expression analysis revealed a significant increase of Axin2, Rn/43, Cdkn3, and/or other Wnt target genes in several distinct cell types (e.g ., AlEnteroPC, TA2, EnteroPrecur). Furthermore, R2M13-26 significantly increased expression of many genes involved in the cell cycle (Table 4) in multiple progenitor cell subtypes in the epithelium, especially the TA2 and injury-specific progenitors (AltEnteroPC).
  • Wnt targets e.g., Ccnbl, Cdca3, Aurka,, Cdkn3
  • the increase in Wnt target gene expression was validated, and an expansion of Axin2 and Cdkn3 expression in the colon crypts of the R2M13-26 treated samples was detected (Figure 27B).
  • the TA1 and TA2 progenitor cells had the highest expression of cell cycle associated genes, and there was an expanded contribution of the R2M13-26 treated samples in these groups at 24-hours after treatment (data not shown), which was consistent with expansion of the progenitors early after dosing.
  • R2M13-26 In addition to increasing expression of genes directly involved in the cell cycle, R2M13-26 also increased expression of several stem/progenitor cell genes such as Lrigl (Powell et al., 2012), Hmga2 (Nishino, Kim, Chada, & Morrison, 2008; Parisi, Piscitelli, Passaro, & Russo, 2020), and Nhp2, a member of the Dyskerin complex associated with telomere maintenance that was shown to be important for stem cell maintenance (Fong, Ho, Inouye, & Tjian, 2014; McCann, Kavari, Burkholder, Phillips, & Hall, 2020).
  • R2M13-26 increased Wnt target and cell cycle gene expression in multiple cell types, predominantly in the different subtypes of stem and progenitor cells including the injury-induced, altered enterocyte cell types, leading to an expansion of the progenitor pool.
  • R2M13-26-treated epithelial cells differentiated more quickly after proliferation.
  • Time-stamping allowed the determination of where the day 6 (48-hour) cells were enriched relative to the day 5 (24-hour) cells for all three treatment conditions, uninjured, injured/anti-GFP and injured/R2M13-26.
  • the day 5 and day 6 uninjured cells were approximately equally represented in all clusters where uninjured cells were present at both timepoints as expected ( Figure 28A, Figure 28B, Figure 28C, and Figure 28D).
  • Figure 28A, Figure 28B, Figure 28C, and Figure 28D Figure 28A, Figure 28B, Figure 28C, and Figure 28D.
  • obvious differences existed between the cell types that were preferentially enriched for the R2M13-26- or anti-GFP-treated day 5 and day 6 injured samples.
  • a reliable standard for validating improved differentiation was that expression of mature, differentiated cell type markers looked more like that of naive, uninjured colon in the R2M13-26 treatment group relative to the anti-GFP controls on day 10 after DSS-induced damage (6-days after R2M13-26 treatment) ( Figure 13). Unlike the anti-GFP treated control samples, R2M13-26-treated samples had recovered enterocytes, goblet cells, enteroendocrine, and tuft cells.
  • mucin and barrier associated gene expression in the R2M13-26-treated samples there was an increase in mucin and barrier associated gene expression in the R2M13-26-treated samples relative to anti-GFP in the TA1 cells.
  • TJP1 ZOl
  • R2M13-26 In addition to its direct impact on epithelial cell regeneration, R2M13-26 also caused a strong increase in expression of genes involved in glutathione (an antioxidant that may play a role in reducing inflammation) conjugation: two glutathione transfersases ( Gstml , Gstm3) and the glutathione peroxidase, Gpx2, all three of which have been reported to be Wnt target genes (Gougelet et al., 2014; Kipp, Banning, & Brigelius-Flohe, 2007).
  • Toxicity study of R2M13-h26 in nonhuman primates (NHP1 [00380] T o evaluate the toxicity of R2M 13 -h26 and to evaluate the potential reversibility of any findings following a 4-week recovery period, a 4-week non-GLP (Good Laboratory Practices) toxicity study of R2M13-h26 following intravenous (IV) bolus injection in Cynomolgus monkeys was performed. In addition, the toxicokinetic (TK) characteristics of R2M13-h26 were determined.
  • TK toxicokinetic
  • Intravenous bolus injections were given once daily to Naive, female, 2-4 year old Cambodian, cynomolgus macaques (2-4 kg) on Days 1, 8, 15, 22, and 29. Vehicle only was used as a control.
  • Clinical pathology hematology, chemistry, coagulation, urinalysis
  • TK sampling was performed at selected time points during doing and to termination; full TK profiles were sampled on Days 1 and 29, and peak/trough on Day 15.
  • Anti-drug antibody (ADA) sampling was performed pre-dose and on Days 15, 29, and 58. Histopathology was performed at termination on Days 30 and 58.
  • Table 11 show the experimental setup of the TK study. Table 11. Dosage used for TK study in NHP
  • Matrix interferences and excess reagents were removed by salt/detergent buffer washes, and the captured complex was subsequently detected by employing a secondary peroxidase-conjugated antibody with specificity to the mouse Fc moiety.
  • the color was developed with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate and HRP reaction was quenched with acidification and the samples were analyzed on a SpectraMax ® Paradigm ® microplate reader.
  • PK Pharmacokinetics
  • Barker Nick Ridgway Rachel A., van Es Johan H., van de Wetering Marc, Begthel Harry, van den Bom Maaike, ... Clevers Hans. (2009). Crypt stem cells as the cells-of-origin of intestinal cancer. Nature, 457(7229), 608-611. https://doi.org/10.1038/nature07602 Barker Nick, van Es Johan H., Kuipers Jeroen, Kujala Pekka, van den Born Maaike,
  • R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/ -catenin signaling. Proceedings of the National Academy of Sciences of the United States of America , 108(28), 11452-11457. https://doi.org/10.1073/pnas.1106083108 Chang, J T. (2020). Pathophysiology of Inflammatory Bowel Diseases. New England
  • Frizzled7 functions as a Wnt receptor in intestinal epithelial Lgr5(+) stem cells. Stem Cell Reports, 4(5), 159-161. https://doi.Org/10.1016/j.stemcr.2015.03.003 Fong, Y. W., Ho, J. J., Inouye, C., & Tjian, R (2014).
  • the dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells. ELife, 3. https://doi.org/10.7554/eLife.03573
  • LGR4 and LGR5 are R-spondin receptors mediating Wnt/p-catenin and Wnt/PCP signalling. EMBO Reports, 72(10), 1055-1061. https://doi.org/10.1038/embor.2011.175
  • Neonatal intestinal organoids as an ex vivo approach to study early intestinal epithelial disorders.
  • H/ACA snoRNA levels are regulated during stem cell differentiation.
  • Nucleic Acids Research, 48(15), 8686-8703. https://doi.org/10.1093/nar/gkaa612 Miao, Ha, de Lau, Yuki, Santos, You, ... Garcia. (2020).
  • Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo. Cell Stem Cell, 27(5), 840-851. https://doi.Org/10.1016/j.stem.2020.07.020 Muhl, L., Genove, G., Leptidis, S., Liu, J., He, L., Mocci, G., ...
  • Betsholtz, C. (2020). Singlecell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination. Nature Communications, 11(1), 3953. https://doi.org/10.1038/s41467-020-17740-l
  • Hmga2 Promotes Neural Stem Cell Self-Renewal in Young but Not Old Mice by Reducing pl6 A I A n A k A 4 A a and pl9 A A A r A f Expression. Cell, 135(2), 227-239. https://doi.Org/10.1016/j.cell.2008.09.017
  • edgeR a Bioconductor package for differential expression analysis of digital gene expression data.
  • Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature, 469(7330), 415-418. https ://doi . org/ 10.1038/nature09637
  • telomeres are an important source of Wnts that supports intestinal crypts. Nature, 557(7704), 242-246. https://doi.org/10.1038/s41586-018-0084-4
  • CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury. Proceedings of the National Academy of Sciences of the United States of America, 114(4), E506-E513. https ://doi . org / 10.1073/pnas.1620059114
  • Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice ELife, 8. https://doi.org/10.7554/eLife.46134
  • Gastroenterology 132(4), 1331-1343 https://doi.Org/10.1053/j.gastro.2007.02 001

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Abstract

La présente divulgation concerne des agonistes de WNT modifiés et des méthodes de traitement de troubles gastro-intestinaux avec des modulateurs de la voie de signalisation de WNT.
EP22767927.1A 2021-03-10 2022-03-09 Modulation de la signalisation wnt dans des troubles gastro-intestinaux Pending EP4305070A1 (fr)

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TW202302647A (zh) 2023-01-16
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